Spire2 and Rab11a synergistically activate myosin-5b motor function.

Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles.

A new C19-diterpenoid alkaloid in Aconitum georgei Comber.

Nine diterpenoid alkaloids were isolated from Aconitum georgei Comber belonging to the genus Aconitum in Ranunculaceae family. Their structures were determinated by using HR-ESI-MS and 1 D/2D NMR spectra as geordine (1), yunaconitine (2), chasmanine (3), crassicauline A (4), forestine (5), pseudaconine (6), 14-acetylalatisamine (7), austroconitine B (8), and talatisamine (9). Among them, compound 1 is a previously undescribed aconitine-type C19-diterpenoid alkaloid, and compounds 3, and 5-9 have not previously been isolated from this species. The results of in vitro experiments indicated that new compound 1 possesses mild anti-inflammatory activity, which inhibited the production of NO in LPS-activated RAW 264.7 cells with an inhibition ratio of 29.75% at 50 µM.

H2 S works synergistically with rhizobia to modify photosynthetic carbon assimilation and metabolism in nitrogen-deficient soybeans.

Hydrogen sulfide (H2 S) performs a crucial role in plant development and abiotic stress responses by interacting with other signalling molecules. However, the synergistic involvement of H2 S and rhizobia in photosynthetic carbon (C) metabolism in soybean (Glycine max) under nitrogen (N) deficiency has been largely overlooked. Therefore, we scrutinised how H2 S drives photosynthetic C fixation, utilisation, and accumulation in soybean-rhizobia symbiotic systems. When soybeans encountered N deficiency, organ growth, grain output, and nodule N-fixation performance were considerably improved owing to H2 S and rhizobia. Furthermore, H2 S collaborated with rhizobia to actively govern assimilation product generation and transport, modulating C allocation, utilisation, and accumulation. Additionally, H2 S and rhizobia profoundly affected critical enzyme activities and coding gene expressions implicated in C fixation, transport, and metabolism. Furthermore, we observed substantial effects of H2 S and rhizobia on primary metabolism and C-N coupled metabolic networks in essential organs via C metabolic regulation. Consequently, H2 S synergy with rhizobia inspired complex primary metabolism and C-N coupled metabolic pathways by directing the expression of key enzymes and related coding genes involved in C metabolism, stimulating effective C fixation, transport, and distribution, and ultimately improving N fixation, growth, and grain yield in soybeans.

Macrostines A and B: Tetracyclic fisicoccane from the fungus Periconia macrospinosa WTG-10.

Two previous unreported fusicoccane diterpenoids macrostines A and B, together with seven known compounds were isolated from an extract of the fungus Periconia macrospinosa WTG-10. Their structures were elucidated by detailed analysis of spectroscopic data, NMR calculations with DP4+, and their absolute configurations were further determined by quantum chemical calculations of ECD spectra or X-crystallography. Macrostines A and B showed no cytotoxicity, antimicrobial activity and inhibitory effect on nitric oxide production in LPS-activated RAW264.7 macrophages. Compound 9 showed moderate activity against Bacillus subtilis.

Polyketides with antimicrobial activities from Penicillium canescens DJJ-1.

Two undescribed polyketides canecines A-B, one unreported cyclopentenone canecine C, together with 12 known compounds were isolated from an extract of the fungus Penicillium canescens DJJ-1. Their structures were elucidated by detailed analysis of spectroscopic data, NMR calculations with dJ-DP4 or DP4+, and their absolute configurations were further determined by quantum chemical calculations of ECD spectra or X-crystallography. Canecine A was a grisan polyketide featuring a dimethyltetrahydro-4H-furo[2,3-b]pyran. Canecine A exhibited significant inhibitory activity against Candida albicans with an MIC value of 1 µg/mL and showed inhibitory effect on nitric oxide production in LPS-activated RAW264.7 macrophages. These results enrich the structural diversities of polyketides from endophytic fungi.

Insect Sf9 cells are suitable for functional expression of insect, but not vertebrate, striated muscle myosin.

Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.

Investigations of specialised metabolites of endophyte Diaporthe destruens hosted in Illigera orbiculata C. Y. Wu.

A chemical investigation of the endophytic fungus Diaporthe destruens from the Hernandiaceae plant Illigera orbiculata C. Y. Wu collected from southern Yunnan Province, China, led to the isolation of six undescribed compounds, including two azaphilone analogs, which are a pair of epimers (13R-hydroxy-chermesinone A and 13S-hydroxy-chermesinone A); a pyrrole derivative (1-(4-(methoxymethyl)-1H-pyrrol-3-yl)ethan-1-one); an isoindolone derivative (4-hydroxy-6-methoxyisoindolin-1-one); a benzylbenzene derivative (destruensine A) and a conjectural fragment of polyketide ((2R,4R)-2-(methoxymethyl)pentane-1,4-diol) along with nine known compounds. Their structures were elucidated by spectroscopic methods and HRESIMS, and the absolute configurations were further confirmed by electronic circular dichroism (ECD) and chemical derivatization. The antimicrobial activities, anti-acetylcholinesterase activities, antiproliferation, and NO production inhibitory effects of compounds 1-15 were evaluated.

A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi.

The complex morphological structure of Aspergillus niger influences its production of proteins, metabolites, etc., making the genetic manipulation and clonal purification of this species increasingly difficult, especially in aconidial Aspergillus niger. In this study, we found that N-acetyl-D-glucosamine (GlcNAc) could induce the formation of spore-like propagules in the aconidial Aspergillus niger SH2 strain. The spore-like propagules possessed life activities such as drug resistance, genetic transformation, and germination. Transcriptomic analysis indicated that the spore-like propagules were resting conidia entering dormancy and becoming more tolerant to environmental stresses. The Dac1 gene and the metabolic pathway of GlcNAc converted to glycolysis are related to the formation of the spore-like propagules, as evidenced by the CRISPRi system, qPCR, and semi-quantitative RT-PCR. Moreover, a method based on the CRISPR-Cas9 tool to rapidly recycle screening tags and recover genes was suitable for Aspergillus niger SH2. To sum up, this suggests that the spore-like propagules are resting conidia and the mechanism of their formation is the metabolic pathway of GlcNAc converted to glycolysis, particularly the Dac1 gene. This study can improve our understanding of the critical factors involved in mechanisms of phenotypic change and provides a good model for researching phenotypic change in filamentous fungi.

The LKB1-like Kinase Elm1 Controls Septin Hourglass Assembly and Stability by Regulating Filament Pairing.

Septins form rod-shaped hetero-oligomeric complexes that assemble into filaments and other higher-order structures, such as rings or hourglasses, at the cell division site in fungal and animal cells [1-4] to carry out a wide range of functions, including cytokinesis and cell morphogenesis. However, the architecture of septin higher-order assemblies and their control mechanisms, including regulation by conserved kinases [5, 6], remain largely unknown. In the budding yeast Saccharomyces cerevisiae, the five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) localize to the bud neck and form an hourglass before cytokinesis that acts as a scaffold for proteins involved in multiple processes as well as a membrane-diffusible barrier between the mother and developing bud [7-9]. The hourglass is remodeled into a double ring that sandwiches the actomyosin ring at the onset of cytokinesis [10-13]. How septins are assembled into a highly ordered hourglass structure at the division site [13] is largely unexplored. Here we show that the LKB1-like kinase Elm1, which has been implicated in septin organization [14], cell morphogenesis [15], and mitotic exit [16, 17], specifically associates with the septin hourglass during the cell cycle and controls hourglass assembly and stability, especially for the daughter half, by regulating filament pairing and the functionality of its substrate, the septin-binding protein Bni5. This study illustrates how a protein kinase regulates septin architecture at the filament level and suggests that filament pairing is a highly regulated process during septin assembly and remodeling in vivo.

The cargo adaptor proteins RILPL2 and melanophilin co-regulate myosin-5a motor activity.

Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.

Regulation of Myosin-5b by Rab11a and the Rab11 family interacting protein 2.

Mammalian myosin-5b (Myo5b) plays a critical role in the recycling of endosomes to the plasma membrane via the interactions with Rab11a and the Rab11 family interacting protein 2 (FIP2). However, it remains unclear on how Rab11a and FIP2 are coordinated in tethering Myo5b with the vesicles and activating the motor function of Myo5b. In the present study, we show that Rab11a binds to the globular tail domain (GTD) of Myo5b and this binding abolishes the head-GTD interaction of Myo5b, thus activating the motor function of Myo5b. On the other hand, FIP2 directly interacts with both Rab11a and the tail of Myo5b, and the binding of FIP2 to Myo5b does not affect Myo5b motor function. Moreover, Rab11a displays higher affinity to FIP2 than to Myo5b, suggesting that Rab11a binds preferentially to FIP2 than to Myo5b. Based on the current findings, we propose that the association of Myo5b with vesicles is mediated by FIP2, which bridges Myo5b and the membrane-bound Rab11a, whereas the motor function of Myo5b is regulated by Rab11a.

Regulation of class V myosin.

Class V myosin (myosin-5) is a molecular motor that functions as an organelle transporter. The activation of myosin-5's motor function has long been known to be associated with a transition from the folded conformation in the off-state to the extended conformation in the on-state, but only recently have we begun to understand the underlying mechanism. The globular tail domain (GTD) of myosin-5 has been identified as the inhibitory domain and has recently been shown to function as a dimer in regulating the motor function. The folded off-state of myosin-5 is stabilized by multiple intramolecular interactions, including head-GTD interactions, GTD-GTD interactions, and interactions between the GTD and the C-terminus of the first coiled-coil segment. Any cellular factor that affects these intramolecular interactions and thus the stability of the folded conformation of myosin-5 would be expected to regulate myosin-5 motor function. Both the adaptor proteins of myosin-5 and Ca2+ are potential regulators of myosin-5 motor function, because they can destabilize its folded conformation. A combination of these regulators provides a versatile scheme in regulating myosin-5 motor function in the cell.

The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function.

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.

None detectable retrograde transport of Chinese botulinum toxin type A in mice by single intramuscular injection.

Botulinum toxin type A (BoNT/A) can specifically cleave synaptosomal associated protein of 25 kDa (SNAP-25) into cleaved SNAP-25 (cl.SNAP-25), thus blocking the synaptic transmission in motor end plate and resulting in paralysis. It has been widely applied in clinical for treatment of various conditions characterized by muscle hyperactivity, such as dystonia and spasticity. BoNT/A is used locally, with little diffusion. Its paralyzing role is considered to be restricted to the nerve muscle junction, or close to the injection site. Recently, more and more studies, however, have suggested that BoNT/A also has central effects. In addition, some investigators have demonstrated that BoNT/A enters into central nervous system via retrograde transport after local intramuscular administration. The retrograde axonal transport of Chinese BoNT/A (CBoNT/A) was studied in this paper, which was rare in report. And the results showed that cl.SNAP-25 appeared not only at the injection site but also in contralateral muscle. Retrograde transport, however, was non-existent or too little to be detected in our study.

Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a.

The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca(2+) or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca(2+), little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function.

The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11.

In the Drosophila melanogaster compound eye, myosin-5 (DmM5) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. However, little is known of how the motor function of DmM5 is regulated at the molecular level. In the present study, we overexpressed DmM5 in Sf9 insect cells and investigated its regulation using purified proteins. We found that the actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid (Ltd) and dRab11 respectively. We found that GTP-bound dRab11, but not Ltd, significantly activates DmM5 actin-activated ATPase activity. Moreover, we identified Gln(1689) in the GTD as the critical residue for the interaction with dRab11 and activation of DmM5 motor function by dRab11. Based on those results, we propose that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.

[Pollution characteristics and distribution of polycyclic aromatic hydrocarbons and organochlorine pesticides in groundwater at Xiaodian Sewage Irrigation Area, Taiyuan City].

Sewage irrigation has been widely used in areas of water shortage in northern China, and it may introduce organic contaminants into groundwater. To characterize the organic contaminants in groundwater in sewage irrigation area, the Xiaodian sewage irrigation area in Shanxi Province was chosen as the case study area. A total of 16 groundwater samples (13 from shallow aquifer, 3 from deep aquifer) were collected. Polycyclic aromatic hydrocarbons (PAHs) were determined by gas chromatography-mass spectrometry (GC-MS) and organochlorine pesticides (OCPs) were ainalyzed by gas chromatography-electron capture detection (GC-ECD). The results showed that the concentrations of PAHs ranged from 13.98 to 505.89 ng x L(-1) with an average concentration of 115.67 ng x (L)(-1). The 2 and 3 ring-PAHs were the main components, while naphthalene and phenanthrene were most frequently detected. The concentrations of OCPs were in the range of 13.91-103.23 ng x L(-1) with an average concentration of 40.99 ng x L(-1), while alpha-HCH, delta-HCH, o,p'-DDD, Aldrin, Endosulfan-sulfate and HCB were most frequently detected. Overall, shallow aquifers appeared more contaminated with these pollutants than deep aquifers. In the area, the order of the organic contaminants concentration in groundwater was: East Main Channel < Beizhang Drainage < Taiyu Drainage, which indicated the quality of groundwater was influenced by the sewage irrigation.

[Influence of organochlorine pesticides in wastewater on the soil along the channel].

Nine profile soil samples and two sewage water samples were collected from Xiaodian sewage irrigation area in Taiyuan city, concentrations of organochlorine pesticides (OCPs) were determined by the gas chromatography coupled with electron capture detector (GC-ECD) to analyze the influence of the leakage of sewage water. The result shows that OCPs in sewage water were mainly composed of HCHs. Concentrations of DDTs and other organochlorine pesticides were very low or out of the detection limit. Concentrations of sigmaOCPs and HCHs in eight profiles near irrigation channels to some extend decreased with the increasing of the linear distance off the channel, which shows influences of the leakage of sewage water on the soil nearby. Concentrations of HCHs clearly decreased with the increasing of soil depth in most profile soils. For the horizontal direction, concentrations of HCHs also decreased with the increasing of the linear distance off the channel. The correlation between HCHs and TOC was positive, but no correlation between pH and HCHs was found.

[Vertical distribution and possible sources of polycyclic aromatic hydrocarbon in sewage area soil].

Nine profile soil samples were collected from Xiaodian sewage irrigation area, Taiyuan city, China. The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were analyzed by gas chromatography equipped with a mass spectrometry detector (GC/ MS). The rank order of the average concentrations of PAHs in the 0-10 cm upper soil layer was background area < swamp area < groundwater irrigation area < sewage irrigation area. The concentrations of PAHs in most profile soils decreased with the increase of depth, and the PAHs were mainly accumulated in the surface soil layer (0-40 cm). 4-6 rings of PAHs were mainly accumulated in the 0-50 cm soil layer, and the accumulation capacity in groundwater-irrigation area was better than that in sewage irrigation area. The correlation between different rings of PAHs and TOC was positive (r(max) = 0.791, P = 0), and the same situation was found for PAHs and sand (r(max) = 0. 882, P = 0). The correlation between PAHs and pH was negative (r(min) = -0.1, P = 0.702). The main source of PAHs in the surface soil layer (0-40 cm) of study area was coal combustion. There were two pollution ways of PAHs in soil, one was settled into soil directly, the other was first settled into water and absorbed on the surface of solid particles, and then got enrichment in soil as irrigation water flew.