[Isolation of human pluripotent mesenchymal stem cells from second-trimester amniotic fluid using two kinds of culture protocol and their differentiation into neuron-like cells].

OBJECTIVE: To isolate mesenchymal stem cells (MSCs) from second-trimester amniotic fluid using an improved two-stage culture protocol, and to induce these MSCs into neuron-like cells. METHODS: An improved two-stage culture protocol for MSCs from amniotic fluid was developed. MSCs from amniotic fluid were induced to differentiate with beta-mercaptoethanol into neuron-like cells. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were employed for analysis of the phenotypic characteristics of the cultured MSCs from amniotic fluid. RESULTS: MSCs from amniotic fluid were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analysis showed that they were positive for CD44, CD29, and CD105, but negative for CD45, CD34, or human leucocyte antigen-DR (HLA-DR). Importantly, a subpopulation of transcription factor Oct-4 positive cells could be detectable in the cultured MSCs from amniotic fluid. Moreover, MSCs from amniotic fluid obtained by two-stage culture protocol showed higher proliferation rate [(15.0+/-2.3)% vs. (10.0+/-1.8)%] and higher Oct-4 positive cell rate [(1.2+/-0.3)% vs. (0.9+/-0.2)%, both P<0.05]. After the induction, MSCs from amniotic fluid displayed processes that formed extensive networks. Positive cells for neurone specific enolase (NSE) constituted (54.76+/-3.65)%, and glial fibrillary acidic protein (GFAP) (36.28+/-4.27)%, respectively. CONCLUSION: We demonstrate that human pluripotent MSCs are present in second-trimester amniotic fluid. The two-stage culture protocol could be a kind of simple one with high performance and it does not interfere with the routine fetal karyotyping. MSCs from amniotic fluid can be induced to differentiate into neuron-like cells in vitro by beta-mercaptoethanol in optimal medium.

Galectin 3 inhibition attenuates renal injury progression in cisplatin-induced nephrotoxicity.

Nephrotoxicity is a major toxic effect in chemotherapy, which constitutes up to 60% of hospitalized acute kidney injury (AKI). Very few treatment options exist to slow the transition from AKI to subsequent chronic kidney diseases (CKD). Here, we demonstrate that galectin-3 (Gal-3), a ß-galactoside binding lectin that plays an important role in kidney fibrosis and renal failure, is one of the key factors for renal injury progression. Ectopic overexpression of Gal-3 significantly decreased the viability of HEK293, simultaneously inducing of cell cycle arrest and apoptosis. However, inhibition of Gal-3, mediated by modified citrus pectin (MCP), predominantly antagonized the pro-apoptotic effects. Mice were pre-treated with normal or 1% MCP-supplemented drinking water 1 week before cisplatin injection. Analyses of serum creatinine and renal tissue damage indicated that MCP-treated mice demonstrated increased renal function and attenuated renal fibrosis after cisplatin-induced injury. MCP-treated mice also demonstrated decreased renal fibrosis and apoptosis, as revealed by masson trichrome staining and Western blot analysis of cleaved caspase-3. Additionally, the protective role of Gal-3 inhibition in the kidney injury was shown to be mediated by protein kinase C α (PKC-α), which promoted cell apoptosis and collagen I synthesis in HEK293 cells. These results demonstrated the potential Gal-3 and PKC-α as therapeutic targets for the treatment of AKI and CKD.

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array.

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H+)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.

Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers.

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wD mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159wD cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.

[Effect of the ethanol extract of Picrorhiza scrophulariiflora on the progression of chronic kidney disease in a rat remnant kidney model].

OBJECTIVE: To investigate the effect of the ethanol extract of Picrorhiza scrophulariiflora (EPS) on renal function and tissue damage in a rat remnant kidney model. METHODS: Rat models of chronic kidney disease induced by 5/6 nephrectomy (5/6 Nx) were randomly assigned into two groups for treatment with a gavage of either EPS or vehicle for 9 weeks. The rats in the control group received only sham operation. RESULTS: Compared with vehicle-treated 5/6 Nx rats, the EPS-treated rats displayed significantly decreased urinary excretion of malondialdehyde, serum levels of AGEs and AOPPs, and increased serum SeGSHPx activities. These changes were associated with attenuated urinary protein excretion, glomerular sclerosis and interstitial fibrosis. CONCLUSION: EPS can obviously improve the renal functions and renal pathologies in rats with chronic kidney disease probably by inhibiting the oxidative stress.

Genetic and biochemical studies in yeast reveal that the cotton fibre-specific GhCER6 gene functions in fatty acid elongation.

3-ketoacyl-CoA synthase catalyses the initial condensation reaction during fatty acid elongation using malonyl-CoA and long-chain acyl-CoA as substrates. Previously, it was reported that several genes encoding putative cotton 3-ketoacyl-CoA synthases were significantly up-regulated during early cotton fibre development. In this study, GhCER6 cDNA that contains an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fibre cells. When GhCER6 was transformed to the Saccharomyces cerevisiae elo3 deletion mutation strain that was deficient in the production of 26-carbon fatty acids and displayed a very slow-growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells. Further, heterologous expression of GhCER6 restored the viability of the S. cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced very-long-chain fatty acids that are essential for cell growth. The results suggest that GhCER6 encodes a functional 3-ketoacyl-CoA synthase.

Advanced oxidation protein products accelerate renal fibrosis in a remnant kidney model.

Accumulation of plasma advanced oxidation protein products (AOPP) has been found in patients with chronic kidney disease. However, the biologic consequences of AOPP consumption on progression of renal disease still are unclear. For testing of the hypothesis that AOPP accelerate progression of chronic kidney disease, Sprague-Dawley rats were subjected to five-sixths nephrectomy (5/6 Nx) or to sham operation. Rats in each group were randomly assigned in three subgroups (n = 30 in each group) and treated with repeated intravenous injections of AOPP-modified rat serum albumin (RSA), unmodified RSA, or vehicle for indicated period. Compared with RSA- or vehicle-treated 5/6 Nx rats, AOPP RSA-treated 5/6 Nx rats displayed greater proteinuria, higher serum creatinine, and lower creatinine clearance. AOPP challenge resulted in more renal hypertrophy, higher macrophage influx, and greater renal fibrosis in the remnant kidney. Chronic administration of AOPP in sham-operated rats increased urinary protein excretion and renal macrophage infiltration, but histologic renal fibrosis was not observed during the study period. AOPP treatment enhanced AOPP level in renal tissue. This was associated with marked increase of thiobarbituric acid reactive substances, decrease of glutathione peroxidase activity, and upregulated expression of monocyte chemoattractant protein-1 and TGF-beta1 in renal cortex. These data indicate that AOPP might be a new and potentially important mediator of renal fibrosis in the remnant kidney. Chronic accumulation of AOPP promotes renal fibrosis probably via a redox-sensitive inflammatory pathway.

Post-translational modifications, but not transcriptional regulation, of major chloroplast RNA-binding proteins are related to Arabidopsis seedling development.

Chloroplast RNA-binding proteins are involved in stabilizing stored chloroplast mRNAs and in recruiting site-specific factors that mediate RNA metabolism. In the present study, we characterized two major chloroplast RNA-binding proteins, cp29A and cp29B, by MALDI-TOF MS, N-terminal sequencing, and ESI-MS/MS following 2D-PAGE separation. Polypeptides derived from cp29A were recovered with free N-terminus or with N-terminal acetylation. In addition to the two isoforms found for cp29A, an isoform derived from cp29B was also observed to have five amino acids cleaved from its N-terminus. Results of quantitative real-time RT-PCR indicate that both genes reached maximal rates of transcription 96 h after commencement of germination and maintained relatively high levels throughout the whole life cycle. Transcription of cp29A and cp29B did not vary significantly under light or dark conditions, although production of the acetylated and N-terminally cleaved protein isoforms exhibited light dependence. Exposure of etiolated Arabidopsis seedlings to light conditions for as short as 9 h restored the modified isoforms to levels similar to those found in green plants. Identification of post-translational modifications in major chloroplast RNA-binding proteins may help elucidate their roles in seedling development and in plant RNA stabilization during the greening process.

Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.

Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes.

An annotation update via cDNA sequence analysis and comprehensive profiling of developmental, hormonal or environmental responsiveness of the Arabidopsis AP2/EREBP transcription factor gene family.

AP2/EREBP transcription factors (TFs) play functionally important roles in plant growth and development, especially in hormonal regulation and in response to environmental stress. Here we reported verification and correction of annotation through an exhaustive cDNA cloning and sequence analysis performed on 145 of 147 gene family members. A RACE analysis performed on genes with potential in-frame up-stream ATG codon resulted in identification of At2g28520 as an authentic AP2/EREBP member and corrected ORF annotations for three other members. A further phylogenetic analysis of this updated and likely complete family divided it into three major subfamilies. The expression patterns of the AP2/EREBP family members among the 11 organ or tissue types were examined using an oligo microarray and their hormonal and environmental responsiveness were further characterized using cDNA custom macroarrays. These detailed expression profile results provide strong support for a role for AP2/EREBP family members in development and in response to environmental stimuli, and a foundation for future functional analysis of this gene family.

Analysis of five differentially expressed gene families in fast elongating cotton fiber.

Using the suppression subtractive hybridization method, we isolated five gene families, including proline-rich proteins (PRPs), arabinogalactan proteins (AGPs), expansins, tubulins and lipid transfer proteins (LTPs), from fast elongating cotton fiber cells. Expression profile analysis using cDNA array technology showed that most of these gene families were highly expressed during early cotton fiber developmental stages (0 C20 days post anthesis, DPA). Many transcripts accumulated over 50-fold in 10 DPA fiber cells than in 0 DPA samples. The entire gene family --AGP, together with 20 individual members in other 4 gene families, are reported in cotton for the first time. Accumulation of cell wall proteins, wall loosening enzymes, microtubules and lipid transfer protein may contribute directly to the elongation and development of fiber cells.