Cytoplasmic ribosomes on mitochondria alter the local membrane environment for protein import.

Most of the mitochondria proteome is nuclear-encoded, synthesized by cytoplasmic ribosomes, and targeted to mitochondria post-translationally. However, a subset of mitochondrial-targeted proteins is imported co-translationally, although the molecular mechanisms governing this process remain unclear. We employ cellular cryo-electron tomography to visualize interactions between cytoplasmic ribosomes and mitochondria in Saccharomyces cerevisiae. We use surface morphometrics tools to identify a subset of ribosomes optimally oriented on mitochondrial membranes for protein import. This allows us to establish the first subtomogram average structure of a cytoplasmic ribosome on the surface of the mitochondria in the native cellular context, which showed three distinct connections with the outer mitochondrial membrane surrounding the peptide exit tunnel. Further, this analysis demonstrated that cytoplasmic ribosomes primed for mitochondrial protein import cluster on the outer mitochondrial membrane at sites of local constrictions of the outer and inner mitochondrial membrane. Overall, our study reveals the architecture and the spatial organization of cytoplasmic ribosomes at the mitochondrial surface, providing a native cellular context to define the mechanisms that mediate efficient mitochondrial co-translational protein import.

The cryo-EM 3D image reconstruction of isolated Lethocerus indicus Z-discs.

The Z-disk of striated muscle defines the ends of the sarcomere, which repeats many times within the muscle fiber. Here we report application of cryoelectron tomography and subtomogram averaging to Z-disks isolated from the flight muscles of the large waterbug Lethocerus indicus. We use high salt solutions to remove the myosin containing filaments and use gelsolin to remove the actin filaments of the A- and I-bands leaving only the thin filaments within the Z-disk which were then frozen for cryoelectron microscopy. The Lethocerus Z-disk structure is similar in many ways to the previously studied Z-disk of the honeybee Apis mellifera. At the corners of the unit cell are positioned trimers of paired antiparallel F-actins defining a large solvent channel, whereas at the trigonal positions are positioned F-actin trimers converging slowly towards their (+) ends defining a small solvent channel through the Z-disk. These near parallel F-actins terminate at different Z-heights within the Z-disk. The two types of solvent channel in Lethocerus are similar in size compared to those of Apis which are very different in size. Two types of α-actinin crosslinks were observed between oppositely oriented actin filaments. In one of these, the α-actinin long axis is almost parallel to the F-actins it crosslinks. In the other, the α-actinins are at a small but distinctive angle with respect to the crosslinked actin filaments. The utility of isolated Z-disks for structure determination is discussed.

Synthesis, crystal structure, cholinesterase inhibitory activity, evaluation of insecticidal activities, and computational studies of new phosphonic acids.

In an attempt to obtain the modified and novel insecticides with low human toxicity, a series of novel mono-, bis-, and tetraphosphonic acid derivatives were designed and characterized by infrared, 1H, 13C, and 31P NMR spectroscopy and X-ray crystallography. The inhibitory effects of the synthesized compounds were evaluated using the in vitro Ellman method on human and insect acetylcholinesterase (AChE). Some of these compounds, which had low human and high insect toxicity, were chosen to assess the killing effects (in vivo) on third larval instar of elm leaf beetle (X. luteola). In vivo and in vitro evidence has revealed that bisphosphonic acids, containing hydrophobic systems, have a good selectivity of insect AChE inhibition. In the present study, docking results showed that bisphosphonic acids had lower binding energy and higher inhibition compared with tetraphosphonic acids due to the type of their topology and the ability of their hydrogen to interact with the catalytic triad (the main active site of the enzyme). Additionally, the QSAR results demonstrated that the major effecting factors on the insecticidal activity of the subject compounds are the hydrophobicity, size, shape, and ability to form a hydrogen bond. The present study can be helpful in the development of new insecticides.

Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution.

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as "curved molecular crystalline layers". Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

The myosin II coiled-coil domain atomic structure in its native environment.

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn't, and skip regions 2 and 4. At the head-tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil "uncoiling" at the head-tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

CryoEM structure of Drosophila flight muscle thick filaments at 7 Å resolution.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin II's long α-helical coiled-coil tail forms the dense protein backbone of filaments, whereas its N-terminal globular head containing the catalytic and actin-binding activities extends outward from the backbone. Here, we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant water bug Lethocerus indicus Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution.

Nanoscale thermal transport across an GaAs/AlGaAs heterostructure interface.

We studied the thermal transport across a GaAs/AlGaAs interface using time-resolved Reflection High Energy Electron Diffraction. The lattice temperature change of the GaAs nanofilm was directly monitored and numerically simulated using diffusive heat equations based on Fourier's Law. The extracted thermal boundary resistances (TBRs) were found to decrease with increasing lattice temperature imbalance across the interface. The TBRs were found to agree well with the Diffuse Mismatch Model in the diffusive transport region, but showed evidence of further decrease at temperatures higher than Debye temperature, opening up questions about the mechanisms governing heat transfer at interfaces between very similar semiconductor nanoscale materials under highly non-equilibrium conditions.

Insights into Actin-Myosin Interactions within Muscle from 3D Electron Microscopy.

Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug Lethocerus sp. The Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states, as well as the fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures as determined by X-ray crystallography needed to explain the structure of the different actomyosin interactions observed in situ.

The influence of oral copper-methionine on matrix metalloproteinase-2 gene expression and activation in right-sided heart failure induced by cold temperature: A broiler chicken perspective.

This study was designed to investigate the expression, activation and activity of matrix metalloproteinase-2 (MMP-2) in the heart of broiler chickens reared in cold conditions and fed with copper-methionine supplement at different levels. The chickens (n=480) were randomly allotted to six treatments and four replicates. Treatments included two rearing temperatures (i.e. normal and cold temperatures) each combined with three levels of supplemental copper-methionine (i.e. 0, 100 and 200mg/kg). On d 38 and 45 of age, four broilers from each treatment were sacrificed and their hearts were stored at -80°C. Right-sided heart failure, as evident from abdominal and pericardial fluid accumulation, was observed in broilers under cold stress and not receiving supplemental copper. This clinical observation was confirmed at molecular level through increased MMP-2 expression, activation and activity in this group. Birds reared under normal temperature, however, were not involved in right-sided heart failure nor benefitted from copper-methionine supplementation. In contrast, gelatin zymography and real-time PCR demonstrated that dietary supplementation with copper-methionine decreased pro-MMP-2 and MMP-2 in the heart of chickens reared in cold conditions. However, gelatin reverse zymography did not show any difference between treatments in tissue inhibitor of metalloproteinase-2. Level of supplementation showed similar effects on parameters determined. It is concluded that dietary supplementation with copper-methionine reduced right-sided heart failure at clinical and molecular levels in cold-stressed chickens.

Expressional and Bioinformatic Analysis of Bovine Filia/Ecat1/Khdc3l Gene: A Comparison with Ovine Species.

Maternal effect genes have highly impressive effects on pre-implantation development. Filia/Ecat1/Khdc3l is a maternal effect gene found in mouse oocytes and embryos, loss of which causes a 50% decrease in fertility. In the present study, we investigated Filia mRNA expression in bovine oviduct, 30- to 40-day fetus, liver, heart, lung, and oocytes (as a positive control), by RT-PCR and detected it only in oocytes. A 443 bp fragment was amplified only in oocytes and was sequenced as a part of bovine predicted Filia mRNA. We analyzed bovine and ovine Filia N-terminal peptide sequence in PHYRE2, and a KH domain was predicted. Protein alignment using ClustalW indicated a highly identical N-terminal extention between the 2 species. Immunohistochemical analysis using anti-bovine Filia antibody showed the expression of Filia protein in the zone surrounding the nuclear membrane, and in the subcortex of ovine oocytes of primary and antral follicles. However, in the bovine, Filia has been found through the oocyte cytoplasm of antral follicles, and here it is further confirmed in the primary follicles. Our data suggests a difference in Filia expression pattern between cow and sheep, although the sequence is highly conserved.

Effects of Dietary Copper-Methionine on Matrix Metalloproteinase-2 in the Lungs of Cold-Stressed Broilers as an Animal Model for Pulmonary Hypertension.

The objective of the present study was to investigate the effects of different levels of copper (as supplemental copper-methionine) on ascites incidence and matrix metalloproteinase-2 (MMP-2) changes in the lungs of cold-stressed broilers. For this purpose, 480 1-day-old Ross 308 broiler chickens were randomly assigned to six treatments. Treatments consisted of two ambient temperatures (thermoneutral and cold stress) each combined with 0, 100, and 200 mg supplemental copper/kg as copper-methionine in a 2 × 3 factorial arrangement in a completely randomized design with four replicates. Ascites was diagnosed based on abdominal and pericardial fluid accumulation at 45 days of age. Fourty-eight broilers were killed at 38 and 45 days of age, and their lungs were collected for biological analysis. Results showed that MMP-2 increased in the lungs of ascitic broilers and that copper-methionine supplementation significantly reduced MMP-2 in cold-stressed broiler chickens. Treatments did not affect tissue inhibitor of metalloproteinase-2 (TIMP-2) at 38 and 45 days of age, and no difference was observed between 100 and 200 mg/kg copper-methionine treatments. In conclusion, copper-methionine at higher than conventional levels of supplementation decreased ascites incidence in low temperature through reduced MMP-2 concentration. Further research is warranted to investigate the effect of copper on MMP-2 concentrations in other tissues with high oxygen demand.

The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst.

Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers.

A chemiluminescent metalloimmunoassay based on copper-enhanced gold nanoparticles for quantification of human growth hormone.

A chemiluminescence (CL) immunoassay was developed to determine human growth hormone (hGH) based on copper-enhanced gold nanoparticles. In this method, gold nanoparticles were deposited on polystyrene wells for adsorption of human growth antibodies as well as catalyst for reducing of copper ions from the copper enhancer solution. The reduction of copper ions was prevented where the gold nanoparticles were covered by the antibody-antigen immunocomplex. The deposited copper on Au nanoparticles was then dissolved in HNO3 solution and quantified using the CL method. The CL intensity response was logarithmically dependent on the hGH concentrations over the range 0.2-50 ng/mL, with a detection limit (3σ) of 0.036 ng/mL.

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode.

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.

Immobilization of specific monoclonal antibody on Au nanoparticles for hGH detection by electrochemical impedance spectroscopy.

A selective and sensitive electrochemical impedance immunosensor based on immobilization of a specific monoclonal antibody on Au nanoparticles modified gold electrode was developed for determination of human growth hormone (hGH). Colloidal gold nanoparticles were coated on the gold electrode through the thiol groups of 1,6-hexanedithiol monolayer as a cross-linker. Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were utilized for characterization of various coating layers. The relative charge transfer resistance of the hGH antibody/Au-colloid/1,6-hexanedithiol modified gold electrode changes linearly with the concentration of the human growth hormone (hGH) solutions. The limit of detection (LOD) was about 0.64 pgmL(-1) and the linear dynamic range was obtained from 3 to 100 pgmL(-1). The affinity of hGH toward immobilized antibody was calculated to be 4.1 x 10(11)M(-1) by the slope of the calibration curve. The proposed immunosensor might be used as an accurate and applicable tool for specific diagnosis and evaluation in paraclinical tests, replacing the routine invasive and hazardous radio-immunoassay (RIA) method.