Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

Differentiation of insulin-producing cells from human neural progenitor cells

BackgroundSuccess in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Methods and FindingsHere we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.ConclusionProduction of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.     

Large scale purification of rapeseed proteins (Brassica napus L.)

Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.     

Molecular mechanisms involved in robustness of yeast central metabolism against null mutations

Adaptive strategies employed by the yeast Saccharomyces cerevisiae provide robustness and adaptability of its central metabolism. Since central metabolism in yeast has been well studied at the enzymatic and genetic levels, it represents an excellent system for evaluating the relative roles of duplicate genes and alternative metabolic pathways as possible mechanisms for the stability of central metabolism against null mutations. Yeast appears to employ a variety of mechanisms to ensure functional robustness of its central metabolism. Uninterrupted flow of energy and precursor metabolites through the pathways of central metabolism via glycolysis (EMP), pentose phosphate shunt (PPS), and the tricarboxylic acid (TCA) cycle are ensured by a variety of adaptive mechanisms. One of the most significant mechanisms appears to be gene duplication events that have produced a number of isozymes functioning under variable environmental and physiological conditions. Alternative pathways represent another important mechanism for increasing the robustness of the system. The robustness of the pathways of central metabolism is apparently higher than that of the other parts of metabolism, because of its exceptional importance to the organism's vitality. The proportion of duplicated viable genes also is substantially larger in central metabolism than that in a pool of other metabolic genes.     

Evidence for mitochondrial gene control of mating types in Phytophthora

When protoplasts carrying metalaxyl-resistant (Mr) nuclei from the A1 isolate of Phytophthora parasitica were fused with protoplasts carrying chloroneb-resistant (Cnr) nuclei from the A2 isolate of the same species, fusion products carrying Mr nuclei were either the A2 or A1A2 type, while those carrying Cnr nuclei were the A1, A2, or A1A2 type. Fusion products carrying Mr and Cnr nuclei also behaved as the A1, A2, or A1A2 type. The result refutes the hypothesis that mating types in Phytophthora are controlled by nuclear genes. When nuclei from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species and vice versa, all of the nuclear hybrids expressed the mating type characteristics of the protoplast parent. The same was true when the nuclei from the A1 isolate of P. parasitica were fused with the protoplasts from the A0 isolate of Phytophthora capsici and vice versa. These results confirm the observation that mating type genes are not located in the nuclei and suggest the presence of mating type genes in the cytoplasms of the recipient protoplasts. When mitochondria from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species, the mating type of three out of five regenerated protoplasts was changed to the A1 type. The result demonstrated the decisive effect of mitochondrial donor sexuality on mating type characteristics of mitochondrial hybrids and suggested the presence of mating type genes in mitochondria. All of the mitochondrial hybrids resulting from the transfer of mitochondria from the A0 isolate of P. capsici into protoplasts from the A1 isolate of P. parasitica were all of the A0 type. The result supports the hypothesis of the presence of mating type genes in mitochondria in Phytophthora.     

Structural characterisation of a highly branched exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2074

Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 when grown in skimmed milk secretes a highly branched exopolysaccharide. The exopolysaccharide has a heptasaccharide repeat unit and is composed of glucose and galactose in the molar ratio 3:4. Using chemical techniques and 1D and 2D NMR spectroscopy the polysaccharide has been shown to possess the following repeat unit structure: [carbohydrate structure: see text].     

Agrin mediates a rapid switch from electrical coupling to chemical neurotransmission during synaptogenesis

In contrast to its well-established actions as an organizer of synaptic differentiation at the neuromuscular junction, the proteoglycan agrin is still in search of a function in the nervous system. Here, we report an entirely unanticipated role for agrin in the dual modulation of electrical and chemical intercellular communication that occurs during the critical period of synapse formation. When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms. Specifically, it led to decreased gap junction-mediated electrical coupling that preceded an increase in nicotinic synaptic transmission. This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases. Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.     

Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.     

Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.