Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.     

Phytoremediation of Pb contaminated soil with polymer-coated EDTA

EDTA-assisted phytoextraction of lead (Pb) has been developed, but concerns have arisen due to the possibility of leaching of both Pb and EDTA to ground water caused by uncontrolled release. We developed five types of controlled-release EDTA (polymer-coated EDTA) by coating the EDTA with a polyolefin polymer. A test of the release rate showed that the duration for the release of 75% of total EDTA ranged from 3 to 210 days. A pot experiment was conducted to compare the effect of these polymer-coated EDTA and non-coated EDTA on the concentrations of Pb and EDTA in soil solution, and Pb accumulation in sorghum (Sorghum bicolor L. cv. EARLY SUMAC) in a Pb-contaminated soil. One of the polymer-coated EDTAs, C-EDTA-4, with a release period of 80 days proved to be the best in decreasing Pb and EDTA concentrations in soil solution, and increasing Pb accumulation in sorghum shoots compared to the direct application of EDTA. Our results suggest that polymer-coated EDTA has a potential for phytoextraction of Pb with a reduced environmental risk.     

Frog atrial natriuretic peptide and cGMP activate amiloride-sensitive Na<Superscript>+</Superscript> channels in urinary bladder cells of Japanese tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

In our previous study, it was suggested that ANP and cGMP may increase Na⁺ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na⁺ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na⁺ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10⁻⁶ M amiloride. These characteristics were similar to those of amiloride-sensitive Na⁺ channels (ENaC). Addition of 10⁻⁹ M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10⁻⁵ M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The addition of ANP failed to activate the ENaC in the presence of 10⁻⁶ M amiloride. These results suggested that ANP and cGMP activate Na⁺ transport via ENaC in the epithelial cells of frog urinary bladder.     

A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation

In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by -1. We also found that U(85) is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U(85) from chemical modification.     

In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation

Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.     

Amino-acid sequence of LC-1 light chain of squid mantle muscle myosin

Tryptic and chymotryptic peptides of the LC-1 light chain of squid mantle muscle myosin were isolated and sequenced by conventional methods, so that the whole amino-acid sequence of the light chain was established. The light chain consisted of 159 amino-acid residues, and its N-terminal alpha-amino group is blocked. Comparing the established sequence with those of vertebrate muscle myosin light chains, only 35% sequence homology was recognized, but a conservative structure was observed in the third domain.     

Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.