Hormonal specificity of the melanotropin-sensitive adenylate cyclase of mouse melanoma and effect of cyclic amp on the tyrosinase activity of mouse melanoma cells,in vitro

Transplantable mouse melanomas possess a melantropin-sensitive adenylate cyclase system which is responsive to α-melanotropin, β-melanotropin, adrenocorticotropin (ACTH) and prostglandin E₁. It was found that sensitivity to ACTH was not directed towrds the ACTH activity but to the intrinsic melanotropin activity of the ACTH molecule. Therefore, the melanotropin-sensitive adenylate cyclase system is hormonally specific to the intrinsic melanotropin activity of peptide hormones and is unique in the melanoma tissue. The significance of the sensitivity to prostaglandin E₁ is obscure at present. The melanotropin-sensitive adenylate cyclase requires the presence of Mg²⁺ or Mn²⁺ for its enzymatic activity. Ca²⁺ inhibit the enzyme in the presence of a wide range of concentrations of Mg²⁺. The enzymic activity is ATP concentration-dependent and the saturation concentration appears to be 1 mM. The enzyme is very labile in the unfractionated tumor homogenates. A washed 11 000 × g particulate fraction, representing about 30–60% of the total enzymic activity, was found to be more stable and could be stored at 5°C for 2 h without appreaciable loss of the activity. This fraction retained sensitivity to melanotropin, prostaglandin E₁ and NaF. About 20% of the activity of the tumor homogenate could not be sedimented by centrifugation at 105 000 × g for 60 min. This “soluble” fraction was not responsive to melanotropin, prostglandin E₁ and NaF and might be a degradative product produced by the fractionation. Cyclic AMP and α-melanotropin were able to increase the tyrosinase activity of isolated mouse melanoma-cells in vitro under the same conditions.     

PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3

Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.     

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ‘‘one amino acid-one codon”-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.     

Influence of drought stress on pistil physiology and reproductive success of two Gossypium hirsutum cultivars differing in drought tolerance

The causes of reproductive failure under drought stress (DS) are poorly understood. We hypothesized that reproductive failure was related to drought-induced changes in pistil biochemistry. To address this hypothesis, a water deficit-induced experiment was conducted with two cotton cultivars (Dexiamian 1, drought tolerant; Yuzaomian 9110, drought sensitive). Results showed that DS decreased the photosynthesis of subtending leaf and downregulated sucrose transporter gene (GhSUT-1) expression in pistil for both cultivars, resulting in lower pistil carbon accumulation which was reflected in the decreased starch accumulation. Lower starch, as potential energy, and adenosine triphosphate (ATP), as direct energy, in droughted pistils suggested less energy for pollen tube entrance into ovules, reducing the fertilized ovule number and fertilization efficiency. Further, although pistil peroxidase activity increased under DS, a higher hydrogen peroxide (H₂O₂) level still was measured in droughted pistils than well-watered pistils, damaging reproductive activities. Moreover, larger decreases in photosynthesis, pistil GhSUT-1 expression, carbon accumulation, starch and ATP contents caused by DS for Yuzaomian 9110 than Dexiamian 1, and different responses of superoxide dismutase and catalase activities, and ascorbic acid and H₂O₂ contents to DS between the two cultivars might be the reasons causing a greater decrease in fertilization efficiency for Yuzaomian 9110 than Dexiamian 1 under DS. Thus, we suggest that decreased ovule fertilization under DS was related to the disorganized carbohydrate metabolism and inefficient antioxidant defense in droughted pistils, and the effects of DS on pistil carbohydrate metabolism and antioxidant defense were more significant for drought-sensitive cultivars than drought-tolerant cultivars.     

Evaluation of the genetic diversity and population structure of Chinese indigenous horse breeds using 27 microsatellite markers

We determined the genetic diversity and evolutionary relationships among 26 Chinese indigenous horse breeds and two introduced horse breeds by genotyping these animals for 27 microsatellite loci. The 26 Chinese horse breeds come from 12 different provinces. Two introduced horse breeds were the Mongolia B Horse from Mongolia and the Thoroughbred Horse from the UK. A total of 330 alleles were detected, and the expected heterozygosity ranged from 0.719 (Elenchuns) to 0.780 (Dali). The mean number of alleles among the horse breeds ranged from 6.74 (Hequ) to 8.81 (Debao). Although there were abundant genetic variations found, the genetic differentiation was low between the Chinese horses, which displayed only 2.4% of the total genetic variance among the different breeds. However, genetic differentiation (pairwise FST) among Chinese horses, although moderate, was still apparent and varied from 0.001 for the Guizou–Luoping pair to 0.064 for the Jingjiang–Elenchuns pair. The genetic differentiation patterns and genetic relationships among Chinese horse breeds were also consistent with their geographical distribution. The Thoroughbred and Mongolia B breeds could be discerned as two distinct breeds, but the Mongolia B horse in particular suffered genetic admixture with Chinese horses. The Chinese breeds could be divided into five major groups, i.e. the south or along the Yangtze river group (Bose, Debao, Wenshan, Lichuan, Jianchang, Guizhou, Luoping, Jinjiang and Dali), the Qinghai‐Tibet Plateau group (Chaidamu, Hequ, Datong, Yushu, Tibet Grassland and Tibet Valley), the Northeast of China group (Elenchuns, Jilin and Heihe), the Northwest of China group (Kazakh, Yili and Yanqi) and the Inner Mongolia group (Mongolia A, Sanhe, Xinihe,Wuzhumuqin and Sengeng). This grouping pattern was further supported by principal component analysis and structure analysis.     

Docking and molecular dynamics studies on the interaction of four imidazoline derivatives with potassium ion channel (Kir6.2)

Kir6.2, a key component of the ATP-sensitive potassium channel (K_ATP), can directly interact with imidazoline derivatives – a kind of potential antidiabetic drug. This paper explores the interaction of Kir6.2 with four imidazoline derivatives by using AutoDock and Gromacs programs. The docking results reveal that the binding sites of the four imidazolines are different from each other: Idazoxan lies in a polar active pocket formed by residues H177, G299 and R301; while RX871024 is situated in a hydrophobic pocket composed of residues F168, M169 and I296; as for Efaroxan and Clonidine, residues H175, R177, K67 and W68 constitute the binding pocket. Based on the docking results, the four imidazoline/Kir6.2 complexes with explicit water and infinite lipid bilayer membrane were constructed to perform molecular dynamics (MD) simulation. The results of MD calculation are as follows: dioleoyl phosphatidyl choline bilayer membrane stabilised the structure of these complexes through polar and nonpolar interaction; Idazoxan and RX871024 are stably combined with Kir6.2 in their docking sites; Efaroxan has a minor change in contrast to the docking result; whereas Clonidine has an obvious change compared to docking conformation. The binding sites and interaction modes of these imidazolines with Kir6.2 may provide theoretical support in the pharmacological study of imidazoline drugs.     

Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation

Inserting foreign epitopes to hepatitis B core (HBc) virus‐like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost‐effective purification process was developed for two chimeric HBc VLPs displaying Epstein–Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core‐HBc was found to be less stable in water environment compared with EBNA1‐HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α‐helix of HCV core‐HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost‐effective purification approach.