Curcumin-Attenuated TREM-1/DAP12/NLRP3/Caspase-1/IL1B, TLR4/NF-κB Pathways, and Tau Hyperphosphorylation Induced by 1,2-Diacetyl Benzene: An in Vitro and in Silico Study.

We aimed to evaluate the effects of 1,2-diacetylbenzene (DAB) and curcumin on neuroinflammation induced by DAB via triggering receptor expressed on myeloid cells 1 (TREM-1), Toll-like receptor 4 (TLR4), and NLR family pyrin domain containing 3 (NLP3)/calcium-dependent activator protein for secretion 1 (CAPS1)/interleukin 1 beta (IL1B) pathways; tau hyperphosphorylation; reactive oxygen species (ROS); and advanced glycation end-product (AGE) in microglia cells; and explore the molecular mechanisms involved in the key genes induced by DAB and targeted by curcumin in silico analysis. In this study, Western blot, quantitative polymerase chain reaction, and immunocytochemistry were used as the key methods in vitro. In silico analysis, GeneMANIA, ToppFun feature, Metascape, CHEA3, Cytoscape, Autodock, and MIENTURNET were the core approaches used. Curcumin inhibited both the DAB-induced TREM-1/DAP12/NLRP3/caspase-1/IL1B pathway and the TLR4/NF-κB pathway. In BV2 cells, curcumin inhibited ROS, AGE, hyperphosphorylation, glycogen synthase kinase-3ß (GSK-3ß), and ß-amyloid while activating nuclear factor erythroid 2-related factor 2 (Nrf2) expression. In silico studies showed that tumor necrosis factor (TNF), IL6, NFKB1, IL10, and IL1B, as well as MTF1 and ZNF267, were shown to be important genes and transcription factors in the pathogenesis of cognitive impairment produced by DAB and curcumin. Three significant miRNAs (hsa-miR-26a-5p, hsa-miR-203a-3p, and hsa-miR-155-5p) implicated in the etiology of DAB-induced cognitive impairment and targeted by curcumin were also identified. Inflammation and cytokine-associated pathways, Alzheimer's disease, and cognitive impairment were characterized as the most significant biological processes implicated in genes, miRNAs, and transcription factors induced by DAB and targeted by curcumin. Our findings provide new insight into fundamental molecular mechanisms implicated in the pathogenesis of cognitive impairment caused by DAB, particularly the effects of neuroinflammation. Furthermore, this study suggests that curcumin might be a promising therapeutic molecule for cognitive impairment treatment through modulating neuroinflammatory responses.

Molecular mechanism of two functional protein structure changes under 2,3-butanedione-induced oxidative stress and apoptosis effects in the hepatocytes.

Food security has become closely watched with the occurrence of a series of food safety incidents in recent years. The widespread adoption of 2,3-butanedione (BUT), as a food additive, is an unpreventable significant risk factor to food security. Based on this, mouse hepatocyte AML-12 cells and two functional proteins (bovine serum albumin and lysozyme) were utilized as targeted receptors to study the adverse effects of BUT at the cellular and molecular levels. Results suggested that BUT could disrupt the redox balance of AML-12 cells, reducing glutathione (GSH) activity fell to 87.18 %, which cannot offset the production of reactive oxygen species (ROS). Meanwhile, the increasement of lipid peroxidation and malondialdehyde (MDA) levels were observed. The mitochondrial membrane function was also abnormal due to the excessive accumulation of ROS and eventually leads to cell apoptosis and death. At the molecular level, the exposure of BUT could alter the skeleton and secondary structure of bovine serum albumin (BSA) and lysozyme (LYZ), and it could statically quench the intrinsic fluorescence of proteins. The combined experiments confirmed proved the potentially toxic effects of BUT accumulation on the detoxification organ, providing theoretical support for the liver diseases caused by BUT exposure, and a reference for the risk assessment of occupational exposure of BUT.

Bacterial diacetyl suppresses abiotic stress-induced senescence in Arabidopsis.

Premature plant senescence induced by abiotic stresses is a major cause of agricultural losses worldwide. Tools for suppressing stress-induced plant senescence are limited. Here, we report that diacetyl, a natural compound emitted by the plant-beneficial bacterium Bacillus amyloliquefaciens, suppresses abscisic acid -mediated foliar senescence in Arabidopsis thaliana under various abiotic stress conditions. Our results establish diacetyl as an effective protector against stress-induced plant senescence and reveal a molecular mechanism for bacteria-enhanced plant stress resistance.

Effects of extended pharmacological disruption of zebrafish embryonic heart biomechanical environment on cardiac function, morphology, and gene expression.

BACKGROUND: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression. RESULTS: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation. CONCLUSIONS: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified.

The Amyloid Aggregation Accelerator Diacetyl Prevents Cognitive Decline in Alzheimer's Mouse Models.

Diacetyl (DA), a food flavorant, is linked with occupational lung disease. Our in vitro experiments described the formation of a covalent adduct by DA with Arg5 of the Aß1-42 peptide, which resulted in only a transient increase in neurotoxicity in SH-SY5Y cells. However, in vivo implications of these effects on Alzheimer's disease (AD) pathogenesis and the underlying mechanisms remain poorly understood. In the APP/PS1 transgenic AD mouse model, DA treatment did not exacerbate learning and memory deficits in the Morris water maze test. Moreover, DA increased the Aß1-42 plaque burden and decreased neuronal inflammation in the transgenic AD mice. Additionally, cognitive impairment induced by intracerebroventricular Aß1-42 was restored by the DA treatment, as assessed by the T-maze test. A corresponding mitigation of neuronal inflammation was also observed in the hippocampus of these nontransgenic mice due to the acceleration of Aß1-42 aggregation by DA into nontoxic plaques. The data from SDS-PAGE, dot-blot, and TEM in vitro experiments corroborated the acceleration of the Aß1-42 aggregation observed in vivo in AD animal models and characterized the DA-induced formation of Aß1-42 fibrils. Such Aß1-42-DA fibrils were unstable in the presence of detergent and amenable to detection by the thioflavin T reagent, thus underscoring the distinct assembly of these fibrils compared to that of the fibrils of the native Aß1-42. Taken together, the results of this study present for the first time the in vivo implications of the DA-induced acceleration of Aß1-42 and may provide a strategy for the rational design of Aß1-42 aggregation accelerators as AD therapeutics that promote oligomer-free Aß1-42 fibril formation.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Cauliflower mosaic virus P6 inclusion body formation: A dynamic and intricate process.

During an infection, Cauliflower mosaic virus (CaMV) forms inclusion bodies (IBs) mainly composed of viral protein P6, where viral activities occur. Because viral processes occur in IBs, understanding the mechanisms by which they are formed is crucial. FL-P6 expressed in N. benthamiana leaves formed IBs of a variety of shapes and sizes. Small IBs were dynamic, undergoing fusion/dissociation events. Co-expression of actin-binding polypeptides with FL-P6 altered IB size distribution and inhibited movement. This suggests that IB movement is required for fusion and growth. A P6 deletion mutant was discovered that formed a single large IB per cell, which suggests it exhibited altered fusion/dissociation dynamics. Myosin-inhibiting drugs did not affect small IB movement, while those inhibiting actin polymerization did. Large IBs colocalized with components of the aggresome pathway, while small ones generally did not. This suggests a possible involvement of the aggresome pathway in large IB formation.

NPY/NPF-Related Neuropeptide FLP-34 Signals from Serotonergic Neurons to Modulate Aversive Olfactory Learning in Caenorhabditis elegans.

Aversive learning is fundamental for animals to increase chances of survival. In addition to classical neurotransmitters, neuropeptides have emerged to modulate such complex behaviors. Among them, neuropeptide Y (NPY) is well known to promote aversive memory acquisition in mammals. Here we identify an NPY/neuropeptide F (NPF)-related neuropeptide system in Caenorhabditis elegans and show that this FLP-34/NPR-11 system is required for learning negative associations, a process that is reminiscent of NPY signaling in mammals. The Caenorhabditis elegans NPY/NPF ortholog FLP-34 displays conserved structural hallmarks of bilaterian-wide NPY/NPF neuropeptides. We show that it is required for aversive olfactory learning after pairing diacetyl with the absence of food, but not for appetitive olfactory learning in response to butanone. To mediate diacetyl learning and thus integrate the aversive food context with the diacetyl odor, FLP-34 is released from serotonergic neurons and signals through its evolutionarily conserved NPY/NPF GPCR, NPR-11, in downstream AIA interneurons. NPR-11 activation in the AIA integration center results in avoidance of a previously attractive stimulus. This study opens perspectives for a deeper understanding of stress conditions in which aversive learning results in excessive avoidance.SIGNIFICANCE STATEMENT Aversive learning evolved early in evolution to promote avoidance of dangerous and stressful situations. In addition to classical neurotransmitters, neuropeptides are emerging as modulators of complex behaviors, including learning and memory. Here, we identified the evolutionary ortholog of neuropeptide Y/neuropeptide F in the nematode Caenorhabditis elegans, and we discovered that it is required for olfactory aversive learning. In addition, we elucidated the neural circuit underlying this avoidance behavior, and we discovered a novel coordinated action of Caenorhabditis elegans neuropeptide Y/neuropeptide F and serotonin that could aid in our understanding of the molecular mechanisms underlying stress disorders in which excessive avoidance results in maladaptive behaviors.

Bacteria-derived diacetyl enhances Arabidopsis phosphate starvation responses partially through the DELLA-dependent gibberellin signaling pathway.

Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil microorganisms that colonize roots and stimulate plant growth. Some PGPR strains can directly regulate plant growth in the absence of physical contact with the plant, via volatile organic compounds (VOCs) emissions. Recently, we have described that Arabidopsis thaliana respond differentially to diacetyl, a VOC from Bacillus amyloliquefaciens strain GB03 (GB03), through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. Under phosphate deficient conditions, diacetyl enhances salicylic acid- and jasmonic acid-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Here, we show that application of exogenous gibberellin (GA) partially alleviates the deleterious effect caused by either B. amyloliquefaciens GB03 VOCs or diacetyl in Arabidopsis under phosphate deficient conditions, while DELLA quadruple mutant exposed to GB03 VOCs exhibits a partial reduction on the stress symptoms. Moreover, diacetyl appears to enhance DELLA protein accumulation and increase the expression of several GA deactivation-related genes. These findings suggest that the DELLA-mediated GA signaling pathway is involved in the bi-faceted role of GB03 VOCs in regulating plant growth.

Preparation, characterization and comparison of biological potency in two new Zn(II) and Pd(II) complexes of butanedione monoxime derivatives.

A novel Schiff base ligand (2-iminothiophenol-2,3-butanedione monoxime, ITBM) and its complexes with Pd(II) and Zn(II) metal ions ([M(ITBM)2]Cl2) were synthesized and characterized in the present study. The formulated complexes were evaluated for in vitro antioxidant activity as radical scavengers against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•). According to the results, antioxidant activity of Pd complex (IC50=36 mg L-1) was more effective than that of Zn(II) complex (IC50=72 mg L-1). Biophysical techniques along with computational modeling were employed to examine the binding of these complexes with human serum albumin (HSA) as the model protein. The trial findings revealed an interaction between Schiff base complexes and HSA with a modest binding affinity [Kb=6.31(±0.11)×104 M-1 for Zn(II) complex and 0.71(±0.05)×104 M-1 for Pd(II) complex at 310 K]. An intense fluorescence quenching of protein through a static quenching mechanism was occurred due to the binding of both complexes to HSA. Hydrogen bonds and van der Waals forces in both examined systems were the main stabilizing forces in the development of drug-protein complex. Based on far-UV-CD observations, the content of α-helical structure in the protein was reduced through induction by both complexes. Analysis of protein-ligand docking demonstrated binding of the two Schiff base complexes to residues placed in the IIA subdomain of HSA. In addition, Zn complex with HSA showed a stronger binding ability than that of Pd complex.Communicated by Ramaswamy H. Sarma.

Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner.

Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity.

Participation of actin filaments, myosin and phosphatidylinositol 3-kinase in the formation and polarisation of tetraspore germ tube of Gelidium floridanum (Rhodophyta, Florideophyceae).

This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3-kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum. After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6 h. In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F-actin. In the presence of cytochalasin B, an inhibitor of F-actin, latrunculin B, an inhibitor of G-actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical-shaped chloroplasts were observed in the central region with a few F-actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation. It was concluded that F-actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F-actin. Thus, F-actin regulates the polarisation and germination processes of tetraspores of G. floridanum.

Temporally specific engagement of distinct neuronal circuits regulating olfactory habituation in Drosophila.

Habituation is the process that enables salience filtering, precipitating perceptual changes that alter the value of environmental stimuli. To discern the neuronal circuits underlying habituation to brief inconsequential stimuli, we developed a novel olfactory habituation paradigm, identifying two distinct phases of the response that engage distinct neuronal circuits. Responsiveness to the continuous odor stimulus is maintained initially, a phase we term habituation latency and requires Rutabaga Adenylyl-Cyclase-depended neurotransmission from GABAergic Antennal Lobe Interneurons and activation of excitatory Projection Neurons (PNs) and the Mushroom Bodies. In contrast, habituation depends on the inhibitory PNs of the middle Antenno-Cerebral Track, requires inner Antenno-Cerebral Track PN activation and defines a temporally distinct phase. Collectively, our data support the involvement of Lateral Horn excitatory and inhibitory stimulation in habituation. These results provide essential cellular substrates for future analyses of the molecular mechanisms that govern the duration and transition between these distinct temporal habituation phases. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Effects of Diacetyl Flavoring Exposure in Mice Metabolism.

Diacetyl is a flavoring that imparts a buttery flavor to foods, but the use or exposure to diacetyl has been related to some diseases. We investigated the effect of oral intake of diacetyl in male and female C57/Bl mice. We performed a target metabolomics assay using ultraperformance liquid chromatography paired with triple quadrupole mass spectrometry (UPLC-MS/MS) for the determination and quantification of plasmatic metabolites. We observed alterations in metabolites present in the urea and tricarboxylic acid (TCA) cycles. Peroxynitrite plasmatic levels were evaluated by a colorimetric method, final activity of superoxide dismutase (SOD) was evaluated by an enzymatic method, and mouse behavior was evaluated. Majority of the assay showed differences between control and treatment groups, as well as between genders. This may indicate the involvement of sex hormones in the regulation of a normal metabolic profile, and the implication of sex differences in metabolite disease response.

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 µm are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode's solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 °C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

The Broadly Tuned Odorant Receptor OR1A1 is Highly Selective for 3-Methyl-2,4-nonanedione, a Key Food Odorant in Aged Wines, Tea, and Other Foods.

Key food odorants are the most relevant determinants by which we detect, recognize, and hedonically evaluate the aroma of foods and beverages. Odorants are detected by our chemical sense of olfaction, comprising a set of approximately 400 different odorant receptor types. However, the specific receptor activity patterns representing the aroma percepts of foods or beverages, as well as the key food odorant agonist profiles of single-odorant receptors, are largely unknown. We aimed to establish comprehensive key food odorant agonist profiles of 2 unrelated, broadly tuned receptors, OR1A1 and OR2W1, that had been associated thus far with mostly non-key food odorants and shared some of these agonists. By screening both receptors against 190 key food odorants in a cell-based luminescence assay, we identified 14 and 18 new key food odorant agonists for OR1A1 and OR2W1, respectively, with 3-methyl-2,4-nonanedione emerging as the most potent agonist for OR1A1 by 3 orders of magnitude, with a submicromolar half maximal effective concentration. 3-Methyl-2,4-nonanedione has been associated with a prune note in oxidized wine and is an aroma determinant in tea and apricots. Further screening against the entire set of 391 human odorant receptors revealed that 30 or 300 µmol/L 3-methyl-2,4-nonanedione activated only 1 receptor, OR1A1, suggesting a unique role of OR1A1 for the most sensitive detection of this key food odorant in wine, tea, and other food matrices.

[Effect of 2,3-butanedione monoxime on calcium paradox-induced heart injury in rats].

OBJECTIVE: To investigate the Effect of 2,3-butanedione monoxime (BDM) on calcium paradox-induced heart injury and its underlying mechanisms. METHODS: Thirty-two adult male SD rats were randomized into 4 groups, namely the control group, BDM treatment control group, calcium paradox group, and BDM treatment group. Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion and the left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP) were monitored. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Lactate dehydrogenase (LDH) content in the coronary flow was determined. Triphenyltetrazolium chloride staining was used to measure the infarct size, and myocardial cell apoptosis was tested with TUNEL method. Western blotting was used to determine the expression of cleaved caspase-3 and cytochrome c. RESULTS: Compared with the control group, BDM at 20 mmol/L had no effect on cardiac performance, cell death, apoptotic index or the content of LDH, cleaved caspase-3 and cytochrome c at the end of perfusion under control conditions (P>0.05). Calcium paradox treatment significantly decreased the cardiac function and the level of LVDP and induced a larger infarct size (P<0.01), an increased myocardial apoptosis index (P<0.01), and up-regulated expressions of cleaved caspase-3 and cytochrome c (P<0.01). BDM (20 mmol/L) significantly attenuated these effects induced by calcium paradox, and markedly down-regulated the levels of LVEDP and LDH (P<0.01), lowered myocardial apoptosis index, decreased the content of cleaved caspase-3 and cytochrome c (P<0.01), increased LVDP, and reduced the infarct size (P<0.01). CONCLUSION: BDM suppresses cell apoptosis and contracture and improves heart function and cell survival in rat hearts exposed to calcium paradox, suggesting the value of BDM as an potential drug for myocardial ischemia reperfusion injur.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Mitochondrial respiratory inhibition by 2,3-butanedione monoxime (BDM): implications for culturing isolated mouse ventricular cardiomyocytes.

Experiments in isolated ventricular cardiomyocytes have greatly facilitated the study of cellular and subcellular physiology in the heart. However, the isolation and culture of high-quality adult murine ventricular cardiomyocytes can be technically challenging. In most experimental protocols, the culture of viable adult murine cardiomyocytes for prolonged time periods is achieved with the addition of the myosin II ATPase inhibitors blebbistatin and/or 2,3-butanedione monoxime (BDM). These drugs are added to increase cell viability and life span by inhibiting spontaneous cardiomyocyte contraction, thereby preventing calcium overload, cell hypercontracture, and cell death. While the addition of BDM has been reported to prolong the life span of isolated adult murine cardiomyocytes, it is also associated with several off-target effects. Here, we report a novel off-target effect, in which BDM inhibits mitochondrial respiration by acting directly on the electron transport chain to reduce cell viability. In contrast, when cells were cultured with blebbistatin alone, cells survived for longer, and no metabolic off-target effects were observed. Based on these novel observations, we recommend that culture media for isolated mouse ventricular cardiomyocytes should be supplemented with blebbistatin alone, as BDM has the potential to affect mitochondrial respiration and cell viability, effects which may impact adversely on subsequent experiments.

Motion analysis and ultrastructural study of a colonial diatom, Bacillaria paxillifer.

The pennate diatom, Bacillaria paxillifer, forms a colony in which adjacent cells glide smoothly and almost continuously, yet no obvious apparatus driving the movement, such as flagella or cilia, is observed. Thus far, neither the mechanism nor physiological significance of this movement has been well understood. Here, we report quantitative analysis of the gliding motion of B. paxillifer and morphological analysis of this diatom with light and electron microscopes. The gliding of pairs of adjacent B. paxillifer cells in a colony was cyclic with rather constant periods while the average gliding period varied from a few seconds to multiples of 10 s among colonies. The gliding was compromised reversibly by inhibitors for actin and myosin, suggesting involvement of the actomyosin system. Indeed, we observed two closely apposed actin bundles near the raphe by fluorescence-labeled phalloidin staining. Using electron microscopy, we observed filamentous structures that resemble the actin bundles seen with fluorescence microscopy, and we also found novel electron-dense structures located between the plasma membrane and these actin-like filaments. From these and other observations, we suggest that B. paxillifer also uses actin bundles and propose a putative myosin as a molecular motor in the gliding of unicellular diatoms.