A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

Yak lactate dehydgogenase A4: purification, properties, and cDNA cloning

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.     

Fractionation and characterization of biologically-active polysaccharides from Artemisia tripartita

The leaves of Artemisia species have been traditionally used for prevention and treatment of a number of diseases. In this study, five polysaccharide fractions (designated A-I-A-V) were isolated from the leaves of Artemisia tripartita Rydb. by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, and chromatography. The homogeneity and average molecular weight of each fraction were determined by high performance size-exclusion chromatography. Sugar composition analysis revealed that Artemisia polysaccharides consisted primarily of xylose, glucose, arabinose, galactose, and galactosamine. Moreover, all fractions contained at least 3.4% sulfate, and fractions A-II-A-V contained an arabinogalactan type II structure. All fractions exhibited macrophage-activating activity, enhancing production of intracellular reactive oxygen species and release of nitric oxide, interleukin 6, interleukin 10, tumor necrosis factor alpha, and monocyte chemotactic protein 1. In addition, all fractions exhibited scavenging activity for reactive oxygen species generated enzymatically or produced extracellularly by human neutrophils. Finally, fractions A-I and A-V exhibited complement-fixing activity. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Artemisia extracts, and suggest the possibility of using Artemisia polysaccharides as an immunotherapeutic adjuvant.     

Quantification of N2O fluxes from soil–plant systems may be biased by the applied gas chromatograph methodology

With regard to measuring nitrous oxide (N₂O) emissions from biological sources, there are three most widely adopted methods that use gas chromatograph with an electron capture detector (GC–ECD). They use: (a) nitrogen (N₂) as the carrier gas (DN); (b) ascarite as a carbon dioxide (CO₂) trap with DN (DN-Ascarite); and (c) a mixture gas of argon and methane as the carrier (AM). Additional methods that use either a mixture of argon and methane (or of CO₂ and N₂) as a make-up gas with the carrier nitrogen or soda lime (or ascarite) as a CO₂ trap with the carrier helium have also been adopted in a few studies. To test the hypothesis that the use of DN sometimes considerably biases measurements of N₂O emissions from plants, soils or soil–plant systems, experiments were conducted involving DN, AM and DN-Ascarite. When using DN, a significant relationship appeared between CO₂ concentrations and the apparent N₂O concentrations in air samples. The use of DN led to significantly overestimated N₂O emissions from detached fresh plants in static chamber enclosures. Meanwhile, comparably lower emissions were found when using either the DN-Ascarite or AM methods. When an N₂O flux (from a soil or a soil–plant system), measured by DN in combination with sampling from the enclosure of a static opaque chamber, was greater than 200 μg N m^?2 h^?1, no significant difference was found between DN and DN-Ascarite. When the DN-measured fluxes were within the ranges of <?30, ?30–0, 0–30, 30–100 and 100–200 μg N m^?2 h^?1, significant differences that amounted to ?72, ?22, 5, 38 and 64 μg N m^?2 h^?1, respectively, appeared in comparison to DN-Ascarite. As a result, the DN measurements in rice–wheat and vegetable fields overestimated both annual total N₂O emissions (by 7–62%, p?<?0.05) and direct emission factors for applied nitrogen (by 6–65%). These results suggest the necessity of reassessing the available data determined from DN measurements before they are applied to inventory estimation. Further studies are required to explore appropriate approaches for the necessary reassessment. Our results also imply that the DN method should not be adopted for measuring N₂O emissions from weak sources (e.g., with intensities less than 200 μg N m^?2 h^?1). In addition, we especially do not recommend the use of DN to simultaneously measure N₂O and CO₂ with the same ECD.     

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward β-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic β cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.     

The mitochondrial genome of <Emphasis Type="Italic">Protohermes concolorus</Emphasis> Yang et Yang 1988 (Insecta: Megaloptera: Corydalidae)

The first complete mitochondrial genome of dobsonfly Protohermes concolorus Yang et Yang, 1988 (Megaloptera: Corydalidae) was sequenced in this study. The genome was a circular molecule of 15,851 bp containing the typical 37 genes that arranged in the same order as that of the putative ancestor of hexapods. Sequences overlaps were observed between several neighbor genes, which made the genome relatively compact. The tRNA-Ser (GCT) could not be folded into typical secondary structure because its DHU arm was replaced with a simple loop. Six of the 13 protein genes were terminated with a single T adjacent to a downstream tRNA gene in the same strand. The variation of GC content caused the different nucleotide substitution patterns of the protein genes. The genome was AT-biased with a total A + T content of 75.83% which was also demonstrated by the codon usage. The control region was the most AT-rich region with a sub-region of even higher A + T content. Protein genes of two strands presented opposite CG-skew trends which was also reflected by the codon usage. For most of the amino acids, the protein coding sequences did not prefer to use the cognate codons of corresponding tRNAs and the codon usage of the protein genes was not random. The variation of nucleotide substitution patterns of protein genes was significantly correlated with the GC content. The phylogenetic analyses based on all the 13 protein genes showed that Megaloptera was the sister group of other holometabolous insects except Coleoptera.     

A possible mechanism of processive nucleotide and repeat additions by the telomerase

Telomerase is a specialized cellular ribonucleoprotein complex that can synthesize long stretches of a DNA primer by using an intrinsic RNA template sequence. This requires that the telomerase must be able to carry out both nucleotide and repeat additions. Here, based on available structures and experimental data, a model is presented to describe these two addition activities. In the model, the forward movement of the polymerase active site along the template during the processive nucleotide addition is rectified through the incorporation of a matched base, via the Brownian ratchet mechanism. The unpairing of the DNA:RNA hybrid and then repositioning of product 3′-end after each round of repeat synthesis, which are prerequisites for the processive repeat addition, are caused by a force acting on the primer. The force results from the conformational transition of the stem III pseudoknot, which is mechanically induced by the rotation of TERT fingers together with stem IV loop towards the polymerase active site upon a nucleotide binding. Based on the model, the dynamics of processive nucleotide and repeat additions by recombinant Tetrahymena telomerase is studied analytically, which gives good quantitative explanations to the previous experimental results. Moreover, some predicted results are presented. In particular, it is shown that the repeat addition processivity is mainly determined by the difference between the free-energy change required to disrupt the DNA:RNA hybrid and that required to unfold the stem III pseudoknot. A large difference in free energy corresponds to a low repeat addition processivity while a small difference in free energy corresponds to a high repeat addition processivity.     

Phytochrome-mediated growth inhibition of seminal roots in rice seedlings

In rice ( Oryza sativa ) seedlings, continuous white-light irradiation inhibited the growth of seminal roots but promoted the growth of crown roots. In this study, we examined the mechanisms of photoinhibition of seminal root growth. Photoinhibition occurred in the absence of nitrogen but increased with increasing nitrogen concentrations. In the presence of nitrogen, photoinhibition was correlated with coiling of the root tips. The seminal roots were most photosensitive 48–72 h after germination during the 7-day period after germination. White-light irradiation for at least 6 h was required for photoinhibition, and the Bunsen–Roscoe law of reciprocity was not observed. Experiments with phytochrome mutants showed that far-red light was perceived exclusively by phyA, red light was perceived by both phyA and phyB, and phyC had little or no role in growth inhibition or coiling of the seminal roots. These results also suggest that other blue-light photoreceptors are involved in growth inhibition of the seminal roots. Fluence-response curve analyses showed that phyA and phyB control very low-fluence response and low-fluence response, respectively, in the seminal roots. This was essentially the same as the growth inhibition previously observed at the late stage of coleoptile development (80 h after germination). The photoperceptive site for the root growth inhibition appeared to be the roots themselves. All three phytochrome species of rice were detected immunochemically in roots.