Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage,blood outgrowth endothelial cells and cells from milk teeth

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.     

Mutation c.359_363delGTATTinsATAC in the COL4A5 Causes alport syndrome in a Chinese family

The X-linked form of Alport syndrome is associated with mutations in the COL4A5 gene, which is located at Xq22.3 and encodes the α5 chain of type IV collagen. Here we clinically characterized a Chinese family with Alport Syndrome, but no ocular or hearing abnormalities have been observed in any patient in the family. Through Linkage analysis and direct DNA sequencing, a novel complex deletion/insertion mutation c.359_363delGTATTinsATAC in the COL4A5 gene was identified in the family. The mutation was found in all affected family members, but was not present in the unaffected family individuals or the 200 controls. The predicted mutant protein in the family is a truncated protein consisting of only 153 residues. Our report for the first time revealed that the frameshift mutation in the type IV collagen chain α5 causes only renal disease, without extrarenal lesion. Our study broadens genotypic and phenotypic spectrum of COL4A5 mutations associated with Alport syndrome.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Comparative behavior of Pd and Ag deposition phenomenon on clean α-Al2O3 (001) surface—A first principle study

The nature of bonding at the interface between deposited silver/palladium and clean Al-terminated (001) surface of α-Al₂O₃ has been investigated using a periodic ab initio method. Substantial inter-planar relaxations within the alumina were found at both the interfaces and the bulk. The periodic calculation with both Ag and Pd deposition shows that 10% of loading on alumina results maximum stability. Surface energy and work function calculations were performed to propose the stability for the metals on the studied surfaces. The deposited Ag forms a three-dimensional (3-D) cluster on top of the alumina surface. The Pd cluster formed on the alumina surface is two-dimensional (2-D) and is distorted to accommodate the Ag cluster in its domain. A further low index calculation can explain the reason for a higher stability of the membrane generated over alumina support with silver and palladium. The results are discussed in view of the existing experimental data and models of metal-oxide interface and a reason for the difference of activity of the metal interaction with alumina surface is postulated.     

Estrous Cycle‐Dependent Expression of Estrogen Receptor α in Periodontal Tissue

Estrogen receptor α (ERα) may play important roles in many estrogen physiological effects, but little is known about the fluctuation of ERα during the estrous cycle. In this study, the dynamic expression of ERα mRNA and protein in periodontal tissue during the estrous cycle were examined. Forty 12‐week‐old female rats were divided into four groups, based on the estrous cycle stage, and sacrificed. Immunohistochemistry and in situ hybridization were used to detect dynamic changes in ERα protein and mRNA in periodontal tissue during the estrous cycle, and data were analyzed by one‐way ANOVA and cosinor analysis for temporal patterns. Significant differences (p<0.05) were found in the expression of ERα protein and mRNA among the four groups. The expression of ERα protein and mRNA exhibited an infradian rhythm with a period of about 120 h (five days). The phase and amplitude differences between ERα protein and mRNA were not significant (p>0.05). The results suggest the expression of ERα is dynamic during the estrous cycle and that in the future chronobiologic methods should be used to study the mechanism of estrogen effect on periodontal tissue.     

Differential role of cyclooxygenase-1 and -2 on renal vasoconstriction to α1-adrenoceptor stimulation in normotensive and hypertensive rats

Aims

Hypertension is associated with the impairment of renal cyclooxygenase (COX) activity, which regulates vascular tone, salt and water balance and renin release. We aimed to evaluate the functional role of COX isoforms in kidneys isolated from spontaneously hypertensive rats (SHR) after α₁-adrenoceptor (α₁-AR) stimulation.

Main methods

Male six-month-old SHR and normotensive Wistar-Kyoto rats (WKY) were used. The kidneys were isolated to measure perfusion pressure and COX-1- or COX-2-derived prostanoids in response to α₁-AR activation.

Key findings

The basal perfusion pressure was higher in SHR kidneys compared with WKY kidneys (95 ± 11 vs. 68 ± 6 mm Hg, P < 0.05). Phenylephrine induced a greater vasopressor response in SHR kidneys (EC₅₀ of 1.89 ± 0.58 nmol) than WKY kidneys (EC₅₀ of 3.30 ± 0.54 nmol, P < 0.05 vs. SHR). COX-1 inhibition decreased the α₁-AR-induced vasoconstrictor response in WKY but did not affect SHR response, while COX-2 inhibition diminished the response in SHR. Both basal prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) values were higher in SHR kidney perfusates (P < 0.05) and were reduced by COX-1 and COX-2 inhibitors in both strains. Furthermore, phenylephrine increased PGI₂ through COX-2 in WKY and through COX-1 in SHR, but the agonist did not significantly modify TxA₂ in both strains.

Significance

The data suggest that COX-1contributes to vasoconstrictor effects in WKY kidneys and that COX-2 has the same effect in SHR kidneys. The results also suggest that basal release of COX-2-derived vasoconstrictor prostanoids is involved in renal vascular hypersensitivity in SHR.     

Chemical modification,antioxidant and α-amylase inhibitory activities of corn silk polysaccharides

Water-soluble corn silk polysaccharides (CSPS) were chemically modified to obtain their sulfated, acetylated and carboxymethylated derivatives. Chemical characterization and bioactivities of CSPS and its derivatives were comparatively investigated by chemical methods, gas chromatography, gel filtration chromatography, scanning electron microscope, infrared spectroscopy and circular dichroism spectroscopy, scavenging DPPH free radical assay, scavenging hydroxyl radical assay, ferric reducing power assay, lipid peroxidation inhibition assay and α-amylase activity inhibitory assay, respectively. Among the three derivatives, carboxylmethylated polysaccharide (C-CSPS) demonstrated higher solubility, narrower molecular weight distribution, lower intrinsic viscosity, a hyperbranched conformation, significantly higher antioxidant and α-amylase inhibitory abilities compared with the native polysaccharide and other derivatives. C-CSPS might be used as a novel nutraceutical agent for human consumption.     

Why Are There So Many Diverse Replication Machineries?

The replicon model has initiated a major research line in molecular biology: the study of DNA replication mechanisms. Until now, the majority of studies have focused on a limited set of model organisms, mainly from Bacteria or Opisthokont eukaryotes (human, yeasts) and a few viral systems. However, molecular evolutionists have shown that the living world is more complex and diverse than believed when the operon model was proposed. Comparison of DNA replication proteins in the three domains, Archaea, Bacteria, and Eukarya, have surprisingly revealed the existence of two distinct sets of non-homologous cellular DNA replication proteins, one in Bacteria and the other in Archaea and Eukarya, suggesting that the last universal common ancestor possibly still had an RNA genome. A major puzzle is the presence in eukaryotes of the unfaithful DNA polymerase alpha (Pol α) to prime Okazaki fragments. Interestingly, Pol α is specifically involved in telomere biosynthesis, and its absence in Archaea correlates with the absence of telomeres. The recent discovery of telomere-like GC quartets in eukaryotic replication origins suggests a link between Pol α and the overall organization of the eukaryotic chromosome. As previously proposed by Takemura, Pol α might have originated from a mobile element of viral origin that played a critical role in the emergence of the complex eukaryotic genomes. Notably, most large DNA viruses encode DNA replication proteins very divergent from their cellular counterparts. The diversity of viral replication machineries compared to cellular ones suggests that DNA and DNA replication mechanisms first originated and diversified in the ancient virosphere, possibly explaining why they are so many different types of replication machinerie.