Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.     

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.     

Single intravenous injection of plasmid DNA encoding human paraoxonase-1 inhibits hyperlipidemia in rats

Paraoxonase-1 (PON1, EC 3.1.8.1) is a high-density lipoprotein (HDL)-associated antioxidant enzyme, and its activity correlates negatively with the level of plasma low-density lipoprotein cholesterol (LDL-C) and triglyceridemia (TG). In this study, we examined the therapeutic effect of plasmid DNA containing the human PON1 gene (pcDNA/PON1) in hyperlipidemic model rats. The rats were fed a high-fat and high-cholesterol diet for 25 days to produce a hyperlipidemic animal model. Single intravenous injection of pcDNA/PON1 into model rats prevented dyslipidemia and hepatic lipid accumulation. The mechanisms of pcDNA/PON1 in treating hyperlipidemia were associated with increases of serum antioxidant PON1 and SOD activities, and with reduction of the levels of total cholesterol (TC), LDL-C and TG. The results suggest the potential therapeutic effect of pcDNA/PON1 on hyperlipidemia.     

Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775)

A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forskål, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 0.125 mg/ml. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residue NH₂-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search in protein databases (BLAST) revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. We propose the name Scygonadin (from the gonad of S. serrata) for this antibacterial protein.     

Involvement of apoptosis in malathion-induced cytotoxicity in a grass carp (Ctenopharyngodon idellus) cell line

We investigated the role of apoptosis in malathion-induced cytotoxicity in the grass carp (Ctenopharyngodon idellus) cell line ZC-7901. Fish cells were treated with different concentrations of malathion (0.62-95 mg/L), and the IC(50) ranged from 37.94+/-1.93 mg/L for 12 h to 3.04+/-0.27 mg/L for 72 h by the MTT assay. Apoptosis was detected by confocal laser scanning microscopy, transmission electron microscopy, TUNEL reaction, DNA laddering and a flow cytometric PI staining assay. The results demonstrated that apoptosis was involved in the cytotoxic effect of malathion, and that malathion-induced apoptosis occurred in a dose- and time-dependent manner. In addition, the induction of apoptosis by malathion was accompanied by mitochondrial membrane potential (DeltaPsi(m)) disruption, intracellular Ca(2+) elevation, generation of reactive oxygen species (ROS) and ATP depletion. Our investigation suggested that malathion exerts its cytotoxic effects by the induction of apoptosis via a direct effect on the mitochondria.     

The mitochondrial genome of the chimpanzee louse,Pediculus schaeffi: insights into the process of mitochondrial genome fragmentation in the blood-sucking lice of great apes

Background

Blood-sucking lice in the genera Pediculus and Pthirus are obligate ectoparasites of great apes. Unlike most bilateral animals, which have 37 mitochondrial (mt) genes on a single circular chromosome, the sucking lice of humans have extensively fragmented mt genomes. The head louse, Pediculus capitis, and the body louse, Pe. humanus, have their 37 mt genes on 20 minichromosomes. The pubic louse, Pthirus pubis, has its 34 mt genes known on 14 minichromosomes. To understand the process of mt genome fragmentation in the sucking lice of great apes, we sequenced the mt genome of the chimpanzee louse, Pe. schaeffi, and compared it with the three human lice.

Results

We identified all of the 37 mt genes typical of bilateral animals in the chimpanzee louse; these genes are on 18 types of minichromosomes. Seventeen of the 18 minichromosomes of the chimpanzee louse have the same gene content and gene arrangement as their counterparts in the human head louse and the human body louse. However, five genes, cob, trnS₁, trnN, trnE and trnM, which are on three minichromosomes in the human head louse and the human body louse, are together on one minichromosome in the chimpanzee louse.

Conclusions

Using the human pubic louse, Pt. pubis, as an outgroup for comparison, we infer that a single minichromosome has fragmented into three in the lineage leading to the human head louse and the human body louse since this lineage diverged from the chimpanzee louse ~6 million years ago. Our results provide insights into the process of mt genome fragmentation in the sucking lice in a relatively fine evolutionary scale.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1843-3) contains supplementary material, which is available to authorized users.     

Novel extracellular and nuclear caspase-1 and inflammasomes propagate inflammation and regulate gene expression: a comprehensive database mining study

Background

Caspase-1 is present in the cytosol as an inactive zymogen and requires the protein complexes named “inflammasomes” for proteolytic activation. However, it remains unclear whether the proteolytic activity of caspase-1 is confined only to the cytosol where inflammasomes are assembled to convert inactive pro-caspase-1 to active caspase-1.

Methods

We conducted meticulous data analysis method?s on proteomic, protein interaction, protein intracellular localization, and gene expressions of 114 experimentally identified caspase-1 substrates and 38 caspase-1 interaction proteins in normal physiological conditions and in various pathologies.

Results

We made the following important findings: (1) Caspase-1 substrates and interaction proteins are localized in various intracellular organelles including nucleus and secreted extracellularly; (2) Caspase-1 may get activated in situ in the nucleus in response to intra-nuclear danger signals; (3) Caspase-1 cleaves its substrates in exocytotic secretory pathways including exosomes to propagate inflammation to neighboring and remote cells; (4) Most of caspase-1 substrates are upregulated in coronary artery disease regardless of their subcellular localization but the majority of metabolic diseases cause no significant expression changes in caspase-1 nuclear substrates; and (5) In coronary artery disease, majority of upregulated caspase-1 extracellular substrate-related pathways are involved in induction of inflammation; and in contrast, upregulated caspase-1 nuclear substrate-related pathways are more involved in regulating cell death and chromatin regulation.

Conclusions

Our identification of novel caspase-1 trafficking sites, nuclear and extracellular inflammasomes, and extracellular caspase-1-based inflammation propagation model provides a list of targets for the future development of new therapeutics to treat cardiovascular diseases, inflammatory diseases, and inflammatory cancers.

     

Didymodon liae sp. nov. (Pottiaceae) from Tibet,China

Didymodon liae J. Kou, X.‐M. Shao & C. Feng sp. nov., is described and illustrated from Dagzê County in south central Tibet, China. The new species is most similar to Didymodon australasiae (Hook. & Grev.) R. H. Zander, but differs in plants growing loosely in a thin turf, stems without hyalodermis and sclerodermis, margins recurved from just above the base nearly to the apex, laminal cells strongly bulging, mammillose, covered with simple or forked papillae, and costa with a layer of ventral substereids. The new species is contrasted with other similar species of the genus. Information on its distribution and ecology is also provided.     

Investigation on the Interaction of Hydroxyethyl Starch 130/0.4 (Voluven) and Serum Albumin for Pharmacokinetic and Toxicological Implications

The interaction of hydroxyethyl starch 130/0.4 (Voluven) with human serum albumin (HSA) has been investigated by fluorescence (steady state and synchronous), Fourier transforms infrared (FT‐IR), and circular dichroism (CD) spectroscopies. Analysis of the fluorescence quenching data of HSA by Voluven using the Stern–Volmer method revealed the formation of 1:1 ground‐state complex. Evaluation of binding parameters and binding energy indicated that the binding reaction was exothermic. On the basis of fluorescence measurements, it was concluded that electrostatic forces play a crucial role in stabilizing the complex. The binding distance was calculated by using Förster resonance energy transfer (FRET) theory. The conformational changes of HSA were obtained qualitatively as well as quantitatively using synchronous fluorescence, FT‐IR, and CD. The HSA underwent partial unfolding in the presence of Voluven.