Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept‐source optical coherence tomography

Corneal transplantation by full‐thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high‐quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three‐dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept‐source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude‐scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 μm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 μm and 564.5 ± 12.5 μm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 μm confirmed the biocompatibility through the image analysis of the depth‐intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.      

Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature

When adding beta-glycerophosphate (beta-GP), a weak base, to chitosan aqueous solutions, the polymer remains in solution at neutral pH and room temperature, while homogeneous gelation of this system can be triggered upon heating. It is therefore one of the rare true physical chitosan hydrogels. In this study, physicochemical and rheological properties of chitosan solutions in the presence of acetic acid and beta-GP were investigated as a function of temperature in order to gain a better understanding of the gelation mechanisms. The gel structure formed at high temperature was only partially thermoreversible upon cooling to 5 degrees C because of the existence of remaining associations, confirmed by the spontaneous recovery of the gel after breakup at low temperature. Increasing temperature had no effect on the pH values of this system, while conductivity (and calculated ionic strength) increased. Values from the pH measurements were used to estimate the degree of protonation of each species as a function of temperature. The decreasing ratio of -NH3+ in chitosan and -OPO(O-)2 in beta-GP suggested reduced chitosan solubility along with a diminution of ionic interactions such as ionic bridging with increasing temperature. On the other hand, the increased ionic strength as a function of temperature, in the presence of beta-GP, enhanced screening of electrostatic repulsion and increased hydrophobic effect, resulting in favorable conditions for gel formation. Therefore, our study suggests that hydrophobic interactions and reduced solubility are the main driving force for chitosan gelation at high temperature in the presence of beta-GP.     

Cloning of an aminoalcoholphosphotransferase cDNA from chinese cabbage roots

Aminoalcoholphosphotransferase is the enzyme that catalyzes the synthesis of phosphatidylcholine and phosphatidylethanolamine from diacylglycerol using CDP-aminoalcohol such as CDP-choline and CDP-ethanolamine. To determine its cDNA structure from roots of Chinese cabbage,Brassica campestris L. ssp.pekinensis, degenerate primers were designed from the regions showing high amino acid homology between yeastCPT1 and soybeanAAPT1 and used for PCR amplification of Chinese cabbage DNA. Chinese cabbage aminoalcoholphosphotransferase cDNA (AAPT) contains an open reading frame of 1,167 bp coding for a protein of 389 amino acids. It shared 81% identity and 94% similarity with soybeanAAPT1 at the predicted amino acid level. Hydropathy profile analysis suggested that the predicted protein structure of Chinese cabbage aminoalcoholphosphotransferase was very similar to the soybean enzyme, showing an overall hydrophobicity and having the same number of predicted transmembrane domains. Southern analysis indicated that there might be close isoforms of the enzyme.AAPT was expressed equally well in young shoots and roots.     

Serotoninergic modulation of GABAergic synaptic transmission in developing rat CA3 pyramidal neurons

Serotoninergic modulation of GABAergic mIPSCs was investigated in immature (postnatal 12–16-days old) rat CA3 pyramidal neurons using a conventional whole-cell patch clamp technique. Serotonin or 5-hydroxytryptamine (5-HT) (10 μmol/L) transiently and explosively increased mIPSC frequency with a small increase in the current amplitude. However, 5-HT did not affect the GABA-induced postsynaptic currents, indicating that 5-HT acts presynaptically to facilitate the probability of spontaneous GABA release. The 5-HT action on GABAergic mIPSC frequency was completely blocked by 100 nmol/L MDL72222, a selective 5-HT₃ receptor antagonist, and mimicked by mCPBG, a selective 5-HT₃ receptor agonist. The 5-HT action on GABAergic mIPSC frequency was completely occluded either in the presence of 200 μmol/L Cd²⁺ or in the Na⁺-free external solution, suggesting that the 5-HT₃ receptor-mediated facilitation of mIPSC frequency requires a Ca²⁺influx passing through voltage-dependent Ca²⁺channels from the extracellular space, and that presynaptic 5-HT₃ receptors are less permeable to Ca²⁺. The 5-HT action on mIPSC frequency in the absence or presence of extracellular Na⁺ gradually increased with postnatal development. Such a developmental change in the 5-HT₃ receptor-mediated facilitation of GABAergic transmission would play important roles in the regulation of excitability as well as development in CA3 pyramidal neurons.     

Hydrogen peroxide mediates Rac1 activation of S6K1

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.     

Comparative effect of seeds of Rhynchosia volubilis and soybean on MG-63 human osteoblastic cell proliferation and estrogenicity

The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are caused by a high content of isoflavone, which function as partial agonists or antagonists of estrogen. To compare the estrogenic effects of SRV and soybean on the MG-63 osteoblastic cell proliferation, 70% methanol extracts of SRV or soybean were treated on MG-63 cells. Although biphasic over a concentration range of 0.001 mg/ml-0.1 mg/ml, both SRV and soybean extracts increased MG-63 cell proliferation. However SRV was more effective at increasing the cell proliferation that paralleled with the greater estrogenic effects as determined by estrogen receptor alpha (ERalpha) expression, an estrogenic response element (ERE)-luciferase activity and the selective expression of insulin-like growth factor-I (IGF-I). SRV-induced IGF-I expression resulted from increases in the mRNA levels. Despite the increased expression of ERbeta, ERE activity and IGF-I expression by soybean were lower than those by SRV. Furthermore, the comparable estrogenic effects between SRV and the combined treatment of genistein and daidzein standards at 0.5 x 10(-8) M, which is a concentration of these two isoflavones similar to that of SRV at 0.001 mg/ml, demonstrate that the greater estrogenicity of SRV for MG-63 cell proliferation is mediated by the synergism of low levels of isoflavones for the selective expression of IGF-I.