Cell physiology of cAMP sensor Epac

Many animal studies and human epidemiological findings have shown that impaired growth in utero is associated with physiological abnormalities in later life and have linked this to tissue programming during suboptimal intrauterine conditions at critical periods of development. However, few of these studies have considered the contribution of the placenta to the ensuing adult phenotype. In mammals, the major determinant of intrauterine growth is the placental nutrient supply, which, in turn, depends on the size, morphology, blood supply and transporter abundance of the placenta and on synthesis and metabolism of nutrients and hormones by the uteroplacental tissues. This review examines the regulation of placental nutrient transfer capacity and the potential programming effects of nutrition and glucocorticoid over-exposure on placental phenotype with particular emphasis on the role of the Igf2 gene in these processes.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Molecular cloning and immunolocalization of the 17 kDa myoglobin of Clonorchis sinensis

We purified the 17 kDa protein abundant in Clonorchis sinensis crude extracts. The N-terminal amino acid sequence of this protein was determined and an oligonucleotide probe synthesized. Using this probe, the cDNA encoding the protein was cloned and sequenced from the C. sinensis cDNA library. It was found to consist of a total of 150 amino acids and to have 41% conserved homology with the myoglobin of the trematodes Paramphistomum epiclitum and Isoparorchis hypselobagri. The gene product over-expressed in the bacterial system was purified and identified as the same molecule in the adult worms. BALB/c mouse sera raised against the adult 17 kDa protein revealed that this myoglobin was distributed throughout the parenchymal tissues except for the eggs and reproductive organs and that the protein may be involved in the survival of C. sinensis in the oxygen-depleted environment of the host.     

Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization

Fetal rat skin transitions from scarless fetal-type repair to adult-type repair with scar between day 16 (E16) and day 18 (E18) of gestation (term = 21.5 days). Deficient transforming growth factor (TGF)-beta 1 and -beta 2 injury response has been proposed as a mechanism for scarless fetal-type repair. However, previous fetal studies have inconsistently reported the degree of TGF-beta induction after injury. To minimize developmental variables in fetal versus adult TGF-beta regulation, we narrowed our study to wounded fetal animals. We hypothesize that TGF-beta ligand and receptor expression will be differentially regulated during the transition from early gestation (E16) wounds manifesting scarless fetal-type repair to late gestation (E19) wounds manifesting adult-type repair with scar. In this study, decreased and rapidly cleared TGF-beta 1 and -beta 2 expression accompanied by increased and prolonged TGF-beta 3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGF-beta 1 and -beta 2 expression accompanied by decreased and delayed TGF-beta 3 expression in wounded E19 animals correlated with disorganized collagen architecture. Similarly, expression of TGF-beta receptors type I and II were also increased or prolonged in E19 animals. Our results implicate increased TGF-beta 1, -beta 2, and decreased TGF-beta 3 expression, as well as increased type I and II receptor expression in late gestation fetal scar formation.     

Modification of Si(100) surface by the grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion

The modification of argon plasma-pretreated single-crystal Si(100) wafer surfaces via the UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer (molecular weight approximately 340) for biomaterials applications was explored. The modified Si(100) surfaces were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Surface peroxide concentrations resulting from the argon plasma treatment and subsequent atmospheric exposure were determined by a coupling reaction with diphenylpicrylhydrazyl. The results suggested that a short plasma treatment time of 10 s and brief air exposure were sufficient for generating an optimum amount of peroxides and hydroperoxides for the subsequent UV-induced graft polymerization. The graft concentration of the PEGMA polymer increased with increasing PEGMA macromonomer concentration for the graft polymerization and with increasing UV graft polymerization time. The PEGMA graft-polymerized silicon surface with a high poly(ethylene glycol) graft concentration was very effective in preventing protein adsorption and platelet adhesion. The grafted PEGMA polymer layer on the Si(100) surface exhibited fairly good stability during storage in a buffer solution.     

Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array

Identification of pathogenic Mycobacterium species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two M. avium-intracellulare complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of M. tuberculosis, MAC, M. fortuitum, M. chelonae, M. abscessus, M. kansasii, M. gordonae, M. scrofulaceum, M. szulgai, M. vaccae, M. xenopi, M. terrae, M. flavescens, M. smegmatis, M. malmoense, M. simiae, M. marinum, M. ulcerans, M. gastri, and M. leprae. All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus Mycobacterium. Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for M. marinum and M. ulcerans, which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.     

Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts

Migration of pericytes such as hepatic stellate cells is fundamentally important for diverse biological and pathological processes including tumor invasion and fibrosis. In prototypical migratory cells such as fibroblasts, the small GTPases Rac1 and RhoA govern the assembly of lamellipodia and stress fibers, respectively, cytoskeletal structures that are integral to the cell migration process. The gaseous signaling molecule nitric oxide (NO) influences growth factor chemotactic responses, although this occurs primarily in cell-type-specific ways and through cell biological effects that are poorly characterized. In this study, we use complementary molecular and cell biological approaches to delineate important roles for Rac1, RhoA, and NO in migration of the human hepatic stellate cell line LX2 and primary rat hepatic stellate cells. Both platelet-derived growth factor (PDGF) and Rac1 overexpression drove migration through formation of actin-positive filopodia spikes in LX2 as compared to the formation of lamellipodia in fibroblasts. NO inhibited PDGF- and Rac1-driven migration in LX2 by abrogating filopodia formation and inhibited migration of fibroblasts by attenuating lamellipodial protrusions. Additionally, RhoA conferred resistance to NO inhibition of migration and restored chemotactic responses to PDGF in the absence of functional Rac1 in LX2. In conclusion, these studies identify novel crosstalk between small GTPases, cytoskeletal structures, and NO in pericyte-specific pathways, providing counterbalances in the chemotactic responses to growth factors.     

High density of immobilized galactose ligand enhances hepatocyte attachment and function

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.