Defects in CISD-1, a mitochondrial iron-sulfur protein, lower glucose level and ATP production in Caenorhabditis elegans.

BACKGROUND: CDGSH iron sulfur domain-containing protein 1 (CISD-1) belongs to the CISD protein family that is evolutionary conserved across different species. In mammals, CISD-1 protein has been implicated in diseases such as cancers and diabetes. As a tractable model organism to study disease-associated proteins, we employed Caenorhabditis elegans in this study with an aim to establish a model for interrogating the functional relevance of CISD-1 in human metabolic conditions. METHODS: We first bioinformatically identified the human Cisd-1 homologue in worms. We then employed N2 wild-type and cisd-1(tm4993) mutant to investigate the consequences of CISD-1 loss-of-function on: 1) the expression pattern of CISD-1, 2) mitochondrial morphology pattern, 3) mitochondrial function and bioenergetics, and 4) the effects of anti-diabetes drugs. RESULTS: We first identified C. elegans W02B12.15 gene as the human Cisd-1 homologous gene, and pinpointed the localization of CISD-1 to the outer membrane of mitochondria. As compared with the N2 wild-type worm, cisd-1(tm4993) mutant exhibited a higher proportion of hyperfused form of mitochondria. This structural abnormality was associated with the generation of higher levels of ROS and mitochondrial superoxide but lower ATP. These physiological changes in mutants did not result in discernable effects on animal motility and lifespan. Moreover, the amount of glucose in N2 wild-type worms treated with troglitazone and pioglitazone, derivatives of TZD, was reduced to a comparable level as in the mutant animals. CONCLUSIONS: By focusing on the Cisd-1 gene, our study established a C. elegans genetic system suitable for modeling human diabetes-related diseases.

Energy-Screened Many-Body Expansion: A Practical Yet Accurate Fragmentation Method for Quantum Chemistry.

We introduce an implementation of the truncated many-body expansion, MBE(n), in which the n-body corrections are screened using the effective fragment potential force field, and only those that exceed a specified energy threshold are computed at a quantum-mechanical level of theory. This energy-screened MBE(n) approach is tested at the n = 3 level for a sequence of water clusters, (H2O)N=6-34. A threshold of 0.25 kJ/mol eliminates more than 80% of the subsystem electronic structure calculations and is even more efficacious in that respect than is distance-based screening. Even so, the energy-screened MBE(3) method is faithful to a full-system quantum chemistry calculation to within 1-2 kJ/mol/monomer, even in good quality basis sets such as aug-cc-pVTZ. These errors can be reduced by means of a two-layer approach that involves a Hartree-Fock calculation for the entire cluster. Such a correction proves to be necessary in order to obtain accurate relative energies for conformational isomers of (H2O)20, but the cost of a full-system Hartree-Fock calculation remains smaller than the cost of three-body subsystem calculations at correlated levels of theory. At the level of second-order Møller-Plesset perturbation theory (MP2), a screened MBE(3) calculation plus a full-system Hartree-Fock calculation is less expensive than a full-system MP2 calculation starting at N = 12 water molecules. This is true even if all MBE(3) subsystem calculations are performed on a single 40-core compute node, i.e., without significant parallelization. Energy-screened MBE(n) thus provides a fragment-based method that is accurate, stable in large basis sets, and low in cost, even when the latter is measured in aggregate computer time.

Self-consistent charge embedding at very low cost, with application to symmetry-adapted perturbation theory.

Extended symmetry-adapted perturbation theory (XSAPT) uses a self-consistent charge embedding to capture many-body polarization, in conjunction with a pairwise-additive SAPT calculation of intermolecular interaction energies. The original implementation of XSAPT is based on charges that are fit to reproduce molecular electrostatic potentials, but this becomes a computational bottleneck in large systems. Charge embedding based on modified Hirshfeld atomic charges is reported here, which dramatically reduces the computational cost without compromising accuracy. Exemplary calculations are presented for supramolecular complexes such as C60@C60H28, a DNA intercalation complex, and a 323-atom model of a drug molecule bound to an enzyme active site. The proposed charge embedding should be useful in other fragment-based quantum chemistry methods as well.

Variational Formulation of the Generalized Many-Body Expansion with Self-Consistent Charge Embedding: Simple and Correct Analytic Energy Gradient for Fragment-Based ab Initio Molecular Dynamics.

The many-body expansion (MBE) and its extension to overlapping fragments, the generalized (G)MBE, constitute the theoretical basis for most fragment-based approaches for large-scale quantum chemistry. We reformulate the GMBE for use with embedding charges determined self-consistently from the fragment wave functions, in a manner that preserves the variational nature of the underlying self-consistent field method. As a result, the analytic gradient retains the simple "sum of fragment gradients" form that is often assumed in practice, sometimes incorrectly. This obviates (without approximation) the need to solve coupled-perturbed equations, and we demonstrate stable, fragment-based ab initio molecular dynamics simulations using this technique. Energy conservation fails when charge-response contributions to the Fock matrix are neglected, even while geometry optimizations and vibrational frequency calculations may yet be accurate. Stable simulations can be recovered by means of straightforward modifications introduced here, providing a general paradigm for fragment-based ab initio molecular dynamics.

Accurate and Efficient ab Initio Calculations for Supramolecular Complexes: Symmetry-Adapted Perturbation Theory with Many-Body Dispersion.

Symmetry-adapted perturbation theory (SAPT) provides a chemically meaningful energy decomposition scheme for nonbonded interactions that is useful for interpretive purposes. Although formally a dimer theory, we have previously introduced an "extended" version (XSAPT) that incorporates many-body polarization via self-consistent charge embedding. Here, we extend the XSAPT methodology to include nonadditive dispersion, using a modified form of the many-body dispersion (MBD) method of Tkatchenko and co-workers. Dispersion interactions beyond the pairwise atom-atom approximation improve total interaction energies even in small systems, and for large π-stacked complexes these corrections can amount to several kilocalories per mole. The XSAPT+MBD method introduced here achieves errors of ≲1 kcal/mol (as compared to high-level ab initio benchmarks) for the L7 data set of large dispersion-bound complexes and ≲4 kcal/mol (as compared to experiment) for the S30L data set of host-guest complexes. This is superior to the best contemporary density functional methods for noncovalent interactions, at comparable or lower cost. XSAPT+MBD represents a promising method for application to supramolecular assemblies, including protein-ligand binding.

Understanding the many-body expansion for large systems. III. Critical role of four-body terms, counterpoise corrections, and cutoffs.

Papers I and II in this series [R. M. Richard et al., J. Chem. Phys. 141, 014108 (2014); K. U. Lao et al., ibid. 144, 164105 (2016)] have attempted to shed light on precision and accuracy issues affecting the many-body expansion (MBE), which only manifest in larger systems and thus have received scant attention in the literature. Many-body counterpoise (CP) corrections are shown to accelerate convergence of the MBE, which otherwise suffers from a mismatch between how basis-set superposition error affects subsystem versus supersystem calculations. In water clusters ranging in size up to (H2O)37, four-body terms prove necessary to achieve accurate results for both total interaction energies and relative isomer energies, but the sheer number of tetramers makes the use of cutoff schemes essential. To predict relative energies of (H2O)20 isomers, two approximations based on a lower level of theory are introduced and an ONIOM-type procedure is found to be very well converged with respect to the appropriate MBE benchmark, namely, a CP-corrected supersystem calculation at the same level of theory. Results using an energy-based cutoff scheme suggest that if reasonable approximations to the subsystem energies are available (based on classical multipoles, say), then the number of requisite subsystem calculations can be reduced even more dramatically than when distance-based thresholds are employed. The end result is several accurate four-body methods that do not require charge embedding, and which are stable in large basis sets such as aug-cc-pVTZ that have sometimes proven problematic for fragment-based quantum chemistry methods. Even with aggressive thresholding, however, the four-body approach at the self-consistent field level still requires roughly ten times more processors to outmatch the performance of the corresponding supersystem calculation, in test cases involving 1500-1800 basis functions.

Accuracy of finite-difference harmonic frequencies in density functional theory.

Analytic Hessians are often viewed as essential for the calculation of accurate harmonic frequencies, but the implementation of analytic second derivatives is nontrivial and solution of the requisite coupled-perturbed equations engenders a sizable memory footprint for large systems, given that these equations are not required for energy and gradient calculations in density functional theory. Here, we benchmark the alternative approach to harmonic frequencies based on finite differences of analytic first derivatives, a procedure that is amenable to large-scale parallelization. Not only for absolute frequencies but also for isotopic and conformer-dependent frequency shifts in flexible molecules, we find that the finite-difference approach exhibits mean errors < 0.1 cm-1 as compared to results based on an analytic Hessian. For very small frequencies corresponding to nonbonded vibrations in noncovalent complexes (for which the harmonic approximation is questionable anyway), the finite-difference error can be larger, but even in these cases the errors can be reduced below 0.1 cm-1 by judicious choice of the displacement step size and a higher-order finite-difference approach. The surprising accuracy and robustness of the finite-difference results suggests that availability of the analytic Hessian is not so important in today's era of commodity processors that are readily available in large numbers. © 2017 Wiley Periodicals, Inc.

Understanding the many-body expansion for large systems. II. Accuracy considerations.

To complement our study of the role of finite precision in electronic structure calculations based on a truncated many-body expansion (MBE, or "n-body expansion"), we examine the accuracy of such methods in the present work. Accuracy may be defined either with respect to a supersystem calculation computed at the same level of theory as the n-body calculations, or alternatively with respect to high-quality benchmarks. Both metrics are considered here. In applications to a sequence of water clusters, (H2O)N=6-55 described at the B3LYP/cc-pVDZ level, we obtain mean absolute errors (MAEs) per H2O monomer of ∼1.0 kcal/mol for two-body expansions, where the benchmark is a B3LYP/cc-pVDZ calculation on the entire cluster. Three- and four-body expansions exhibit MAEs of 0.5 and 0.1 kcal/mol/monomer, respectively, without resort to charge embedding. A generalized many-body expansion truncated at two-body terms [GMBE(2)], using 3-4 H2O molecules per fragment, outperforms all of these methods and affords a MAE of ∼0.02 kcal/mol/monomer, also without charge embedding. GMBE(2) requires significantly fewer (although somewhat larger) subsystem calculations as compared to MBE(4), reducing problems associated with floating-point roundoff errors. When compared to high-quality benchmarks, we find that error cancellation often plays a critical role in the success of MBE(n) calculations, even at the four-body level, as basis-set superposition error can compensate for higher-order polarization interactions. A many-body counterpoise correction is introduced for the GMBE, and its two-body truncation [GMBCP(2)] is found to afford good results without error cancellation. Together with a method such as ωB97X-V/aug-cc-pVTZ that can describe both covalent and non-covalent interactions, the GMBE(2)+GMBCP(2) approach provides an accurate, stable, and tractable approach for large systems.

Disruption of the nuclear membrane by perinuclear inclusions of mutant huntingtin causes cell-cycle re-entry and striatal cell death in mouse and cell models of Huntington's disease.

Accumulation of N-terminal fragments of mutant huntingtin (mHTT) in the cytoplasm, nuclei and axons of neurons is a hallmark of Huntington's disease (HD), although how these fragments negatively impact neurons remains unclear. We followed the distribution of mHTT in the striata of transgenic R6/2-J2 HD mice as their motor function declined. The fraction of cells with diffuse, perinuclear or intranuclear mHTT changed in parallel with decreasing motor function. In transgenic mice, medium spiny neurons (MSNs) that exhibited perinuclear inclusions expressed cell-cycle markers typically not seen in the striata of normal mice, and these cells are preferentially lost as disease progresses. Electron microscopy reveals that perinuclear inclusions disrupt the nuclear envelope. The progression of perinuclear inclusions being accompanied by cell-cycle activation and culminating in cell death was also observed in 1° cortical neurons. These observations provide a strong correlation between the subcellular location of mHTT, disruption of the nucleus, re-entry into the cell-cycle and eventual neuronal death. They also highlight the fact that the subcellular distribution of mHTT is highly dynamic such that the distribution of mHTT observed depends greatly on the stage of the disease being examined.

Electronic structure of open-shell tetrahedral {Fe(NO)2}(9) dinitrosyliron complexes.

High-level ab initio excited-state theory is employed to investigate the electronic structure of doublet {Fe(NO)2}(9) species in the ground state and compared with the results obtained by density functional theory. Both of the approaches consistently suggest that the linear NO ligands in dinitrosyliron complexes (DNICs) feature a radical character. Theoretical calculations also predict that the cyanide-supported DNIC anion of [(NC)2Fe(NO)2](-) features C2v symmetry with a Fe-C-N bonding motif, and multireference theories suggest a minimal active space of CAS(9,9) to describe these {Fe(NO)2}(9) compounds, while larger CAS(13,13) calculations do not tend to significantly improve the geometries. Experimental vibration modes of NO ligands are also accurately assigned due to second-order n-electron valence state perturbation theory.

Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy.

Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

Microtubule-associated histone deacetylase 6 supports the calcium store sensor STIM1 in mediating malignant cell behaviors.

Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) storage sensor that promotes cell growth, migration, and angiogenesis in breast and cervical cancers. Here, we report that the microtubule-associated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca(2+) entry (SOCE) between cervical cancer cells and normal cervical epithelial cells. Confocal microscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated α-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacologic inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca(2+) influx, as evidenced by total internal reflection fluorescent images and intracellular Ca(2+) determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypoacetylated α-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior.

The formation stability, hydrolytic behavior, mass spectrometry, DFT study, and luminescence properties of trivalent lanthanide complexes of H2ODO2A.

The trivalent lanthanide complex formation constants (log K(f)) of the macrocyclic ligand H(2)ODO2A (4,10-dicarboxymethyl-1-oxa-4,7,10-triazacyclododecane) have been determined by pH titration techniques to be in the range 10.84-12.62 which increase with increasing lanthanide atomic number, and are smaller than those of the corresponding H(2)DO2A (1,7-dicarboxylmethyl-1,4,7,10-tetraazacyclododecane) complexes. The equilibrium formation of the dinuclear hydrolysis species, e.g. Ln(2)(ODO2A)(2)(µ-OH)(+) and Ln(2)(ODO2A)(2)(µ-OH)(2), dominates over the mononuclear species, e.g. LnODO2A(OH) and LnODO2A(OH)(2)(-). Mass spectrometry confirmed the presence of [Eu(ODO2A)](+), [Eu(ODO2A)(OH)+H](+), [Eu(2)(ODO2A)(2)(OH(2))(2)+H](+), [Eu(ODO2A)(OH)(2)](-) and [Eu(2)(ODO2A)(2)(OH(2))(3)](-) species at pH > 7. Density function theory (DFT) calculated structures of the EuODO2A(H(2)O)(3)(+) and EuDO2A(H(2)O)(3)(+) complexes indicate that three inner-sphere coordinated water molecules are arranged in a meridional configuration, i.e. the 3 water molecules are on the same plane perpendicular to that of the basal N(3)O or N(4) atoms. However, luminescence lifetime studies reveal that the EuODO2A(+) and TbODO2A(+) complexes have 4.1 and 2.9 inner-sphere coordinated water molecules, respectively, indicating that other equilibrium species are also present for the EuODO2A(+) complex. The respective emission spectral intensities and lifetimes at 615 nm (λ(ex) = 395 nm) and 544 nm (λ(ex) = 369 nm) of the EuODO2A(+) and TbODO2A(+) complexes increase with increasing pH, consistent with the formation of µ-OH-bridged dinuclear species at higher pH. Additional DFT calculations show that each Y(iii) ion is 8-coordinated in the three possible cis-[Y(2)(ODO2A)(2)(µ-OH)(H(2)O)(2)](+), trans-[Y(2)(ODO2A)(2)(µ-OH)(H(2)O)(2)](+) and [Y(2)(ODO2A)(2)(µ-OH)(2)] dinuclear complex structures. The first and the second include 6-coordination by the ligand ODO2A(2-), one by the bridged µ-OH ion and one by a water molecule. The third includes 6-coordination by the ligand ODO2A(2-) and two by the bridged µ-OH ions. The two inner-sphere coordinated water molecules in the cis- and trans-[Y(2)(ODO2A)(2)(µ-OH)(H(2)O)(2)](+) dinuclear complexes are in a staggered conformation with torsional angles of 82.21° and 148.54°, respectively.

A dinitrosyliron complex within the homoleptic Fe(NO)4 anion: NO as nitroxyl and nitrosyl ligands within a single structure.

Nitrosylation of the chelate-thiolate-containing dinitrosyliron complex (DNIC) [(S(CH(2))(3)S)Fe(NO)(2)](-) triggers nitric oxide (NO) activation to generate the homoleptic nitrosyl {Fe(NO)(2)}(9) DNIC [Fe(NO)(4)](-) (1) made up of two nitroxyls (or two NO anions) attached to a delocalized {Fe(NO)(2)}(9) motif. The significantly longer N3-O3/N4-O4 [1.380(12) and 1.280(12) Å] and Fe1-N3/Fe1-N4 [2.008(11) and 2.045(10) Ǻ] bond distances reflect that N3-O3 and N4-O4 of complex 1 may act as the nitroxyl-coordinated ligands. That is, the electronic structure of the DNIC 1 is best described as a {Fe(NO)(2)}(9) motif coordinated by two nitroxyl (NO(-)) ligands.

Dissociation kinetics of macrocyclic trivalent lanthanide complexes of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A).

The [H(+)]-catalyzed dissociation rate constants of several trivalent lanthanide (Ln) complexes of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (LnDO2A(+), Ln = La, Pr, Eu, Er and Lu) have been determined in two pH ranges: 3.73-5.11 and 1.75-2.65 at four different temperatures (19-41.0 °C) in aqueous media at a constant ionic strength of 0.1 mol dm(-3) (LiClO(4)). For the study in the higher pH range, i.e. pH 3.73-5.11, copper(II) ion was used as the scavenger for the free ligand DO2A in acetate/acetic acid buffer medium. The rates of Ln(III) complex dissociation have been found to be independent of [Cu(2+)] and all the Ln(III) complexes studied show [H(+)]-dependence at low acid concentrations but become [H(+)]-independent at high acid concentrations. Influence of the acetate ion content in the buffer on the dissociation rate has also been investigated and all the complexes exhibit a first-order dependence on [Acetate]. The dissociation reactions follow the rate law: k(obs) = k(Ac)[Acetate] + K'k(lim)[H(+)]/(1 + K'[H(+)]) where k(AC) is the dissociation rate constant for the [Acetate]-dependent pathway, k(lim) is the limiting rate constant, and K' is the equilibrium constant for the reaction LnDO2A(+) + H(+) ⇔ LnDO2AH(2+). In the lower pH range, i.e. pH 1.75-2.65, the dye indicator, cresol red, was used to monitor the dissociation rate, and all the Ln(III) complexes also show [H(+)]-dependence dissociation pathways but without the rate saturation observed at higher pH range. The dissociation reactions follow the simple rate law: k(obs) = k(H)[H(+)], where k(H) is the dissociation rate constant for the pathway involving monoprotonated species. The absence of an [H(+)]-independent pathway in both pH ranges indicates that LnDO2A(+) complexes are kinetically rather inert. The obtained k(AC) values follow the order: LaDO2A(+) > PrDO2A(+) > EuDO2A(+) > ErDO2A(+) > LuDO2A(+), whereas the k(lim) and k(H) values follow the order: LaDO2A(+) > PrDO2A(+) > ErDO2A(+) > EuDO2A(+) > LuDO2A(+), mostly consistent with their thermodynamic stability order, i.e. the more thermodynamically stable the more kinetically inert. In both pH ranges, activation parameters, ΔH*, ΔS* and ΔG*, for both acetate-dependent and proton-catalyzed dissociation pathways have been obtained for most of the La(III), Pr(III), Eu(III), Er(III) and Lu(III) complexes, from the temperature dependence measurements of the rate constants in the 19-41 °C range. An isokinetic (linear) relationship is found between ΔH* and ΔS* values, which supports a common reaction mechanism.

ZAK negatively regulates RhoGDIbeta-induced Rac1-mediated hypertrophic growth and cell migration.

RhoGDIbeta, a Rho GDP dissociation inhibitor, induced hypertrophic growth and cell migration in a cultured cardiomyoblast cell line, H9c2. We demonstrated that RhoGDIbeta plays a previously undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are blocked by the expression of a dominant-negative form of Rac1. We also demonstrated that knockdown of RhoGDIbeta expression by RNA interference blocked RhoGDIbeta-induced Rac1 expression and cell migration. We demonstrated that the co-expression of ZAK and RhoGDIbeta in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDIbeta-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIbeta.

RhoGDIbeta-induced hypertrophic growth in H9c2 cells is negatively regulated by ZAK.

We found that overexpression of RhoGDIbeta, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIbeta expression by RNA interference blocked hypertrophic growth. We further demonstrated that RhoGDIbeta physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIbeta functions. Moreover, the ZAK-RhoGDIbeta interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIbeta functions through phosphorylation and RhoGDIbeta counteracts the effects of ZAK by physical interaction. Knockdown of ZAK expression in ZAK- and RhoGDIbeta-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIbeta.

Molecular characterization of diapause hormone and pheromone biosynthesis activating neuropeptide from the black-back prominent moth, Clostera anastomosis (L.) (Lepidoptera, Notodontidae).

Using a strategy of rapid amplification of cDNA ends, the cDNA of diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) was cloned from the head of Clostera anastomosis (L.). The Cloan-DH-PBAN cDNA contains an open reading frame encoding a 196-amino acid preprohormone, from which five putative FXPRL peptides, DH, PBAN, alpha-SGNP(SGNP, suboesophageal ganglion neuropeptide), beta-SGNP and gamma-SGNP, are released. Comparing the deduced amino acid sequences from cDNAs of these five FXPRL peptides to those known from other insects, Cloan-DH shows highest similarity of 93.1% to that from Agrotis ipsilon, Cloan-PBAN 93.9% to those from Helicoverpa armigera, Helicoverpa zea and Helicoverpa assulta, which show the highest similarity to species of Noctuidae. Phylogenetic analysis revealed that the Cloan-DH-PBAN gene is relatively closely related to those from Noctuoidea, but distant from those from Tortricoidea, Yponomeutoidea and Bombycoidea species. The DNA sequence encoding Cloan-DH-PBAN was cloned by PCR, which is 3698bp in size and comprises six exons interspersed by five introns. Developmental expression of the DH-PBAN transcripts in the head was also showed by a semi-quantitative RT-PCR method, which was relatively low in larvae and remained low in pupae of both sexes, increased sharply in adults of both sexes.

ZAK re-programs atrial natriuretic factor expression and induces hypertrophic growth in H9c2 cardiomyoblast cells.

Various intracellular or intercellular stimuli have been associated with the development of cardiac cell hypertrophy. However, the mechanisms underlying this association are not completely understood. In a previous study we determined that ZAK mRNA expression is abundant in heart. ZAK is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the stress-activated protein kinase/c-jun N-terminal kinase pathway and activates NF-kappaB. We, therefore, investigated the potential involvement of ZAK (which in cultured H9c2 cardiomyoblast cell is a positive mediator of cell hypertrophy). Our results showed that the expression of a wild-type form of ZAK induces the characteristic hypertrophic growth features, including increased cell size, elevated atrial natriuretic factor expression, and increased actin fiber organization.