Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in?vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.     

Association of heat shock protein 70 with semen quality in boars

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.     

两种土居天牛记要

本文所记两种天牛,在幼虫时期,生活于土内,啮食植物根部,成虫于5,6月间之雨后,出土面交尾产卵。     

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.     

Uroporphyrinogen Decarboxylase as a Potential Target for Specific Components of Traditional Chinese Medicine: A Virtual Screening and Molecular Dynamics Study

Uroporphyrinogen decarboxylase (UROD) has been suggested as a protectant against radiation for head and neck cancer (HNC). In this study, we employed traditional Chinese medicine (TCM) compounds from TCM nawiaT@esabataD (http://tcm.cmu.edu.tw/) to screen for drug-like candidates with potential UROD inhibition characteristics using virtual screening techniques. Isopraeroside IV, scopolin, and nodakenin exhibited the highest Dock Scores, and were predicted to have good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties. Two common moieties, 2H-chromen-2-one and glucoside, were observed among the top TCM candidates. Cross comparison of the docking poses indicated that candidates formed stable interactions with key binding and catalytic residues of UROD through these two moieties. The 2H-chromen-2-one moiety enabled pi-cation interactions with Arg37 and H-bonds with Tyr164. The glucoside moiety was involved in forming H-bonds with Arg37 and Asp86. From our computational results, we propose isopraeroside IV, scopolin, and nodakenin as ligands that might exhibit drug-like inhibitory effects on UROD. The glucoside and 2H-chromen-2-one moieties may potentially be used for designing inhibitors of UROD.     

The triphasic reduction of cytochrome b in the succinate-cytochrome c reductase

In the succinate-cytochrome c reductase, the reduction of cytochrome b has been found to be triphasic: an initial rapid partial reduction was followed first by a rapid oxidation and then finally by a slow reduction. The initial reduction of cytochrome b was faster than that of cytochrome c₁ and the final slow reduction of cytochrome b began when cytochrome c₁ reduction was approaching completion. In presence of the inhibitors antimycin A or HQNO the reduction of cytochrome b became monophasic. Hysteresis or a kinetic cooperative effect of a factor controlling cytochrome b oxidation has been suggested as a possible explanation for the triphasic reduction of cytochrome b.     

Effects of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5'-flanking region of porcine heat shock protein 70.2 gene (HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5'-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype (P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes (P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype (P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype (P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5'-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.     

Comparison of secondary structures of insulin and proinsulin by FTIR

Although the structure of insulin is known in great detail, that of proinsulin has been little investigated, except for a few CD and NMR studies. The secondary structures of human proinsulin are now compared with those of insulin by Fourier Transformed Infrared (FTIR) studies. The deconvolved and second derivative spectra of proinsulin and insulin in the amide I' band region are closely similar with peaks corresponding to -helix, irregular helix, and 3₁₀ helix at nearly identical positions. For both proteins, the relative contents of the above structures as calculated from the peak areas are in good agreement with the values obtained from the known structure of crystalline porcine insulin. However, compared with insulin, proinsulin has markedly more unordered structures as indicated by the area under the peak at 1643.4 cm^–1. In addition, both peak positions and relative areas for turn structure of the prohormone are different from those for insulin. It appears from the above that the A-and B-chain segments of proinsulin and insulin are similar in their secondary structures, especially in helices. The C-chain segment is largely unordered except in a few -turns.     

Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.