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Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency.

Ganoderma lucidum is one of the most famous mushrooms in traditional Chinese medicine. At present, the fully utilized parts of G. lucidum are mainly fruiting body and spore powder. The wild and artificially cultivated G. lucidum fruiting body is costly and rare. Therefore, how to improve the utilization of G. lucidum by means of fermentation is worth investigating. The present study was to perform submerged fermentation of G. lucidum and compare the bioactivities of G. lucidum submerged fermentation broth and fruiting body extract. After the extraction and submerged fermentation methods were optimized, the optimum conditions for extraction were determined as ethanol extraction at 80°C with a solid-to-liquid ratio of 1:30, and those for submerged fermentation were cultivation on malt extract medium for 6 days at 30°C. Under the optimum conditions, the antioxidative activity and tyrosinase inhibition rate of the fermentation broth were 1.2-4.1 fold higher than those of the ethanol extract. Cytotoxicity analysis showed that the ethanol and water extracts and the fermentation broth effectively inhibited pancreatic cancer cells and prostate cancer cells, with much smaller effect on nontumor human embryonic kidney (HEK293T). These results demonstrate that the submerged fermentation could improve the utilization value of G. lucidum and the fermentation broth can be used as an antioxidant additive applied in food, drugs, and cosmetics.

High mobility group box 1 (HMGB1) is an alarmin that may link to obesity and type 2 diabetes mellitus (T2DM). The present study analyzed the correlation between HMGB1/ Toll-like receptor 4 (TLR4) and certain biochemical parameters in obese (OB) diabetic patients. 40 normal glucose tolerant subjects (NGT) and 40 patients with newly diagnosed T2DM were enrolled. All patients were further divided into non-obese NGT (NGT-NOB), obese NGT (NGT-OB), non-obese T2DM (T2DM-NOB) and obese T2DM (T2DM-OB) groups according to body mass index (BMI).The levels of HMGB1 in serum were quantified using ELISA, whereas the mRNA expression levels of TLR4 in peripheral blood mononuclear cells were assessed using reverse transcription-quantitative PCR. The results suggested that the levels of HMGB1 and TLR4 were higher in NGT-OB and T2DM-NOB groups compared with those in NGT-NOB group. Similarly, the levels of these two markers were higher in T2DM-OB group compared with those in NGT-OB group. Correlation analysis indicated that the levels of HMGB1 and TLR4 were positively correlated with triglyceride (TG), fasting plasma glucose (FPG) levels and BMI, whereas a negative correlation between HMGB1 and high density lipoprotein (HDL) was noted. Linear regression analysis suggested that HMGB1 was associated with FPG and TG levels, whereas TLR4 was strongly associated with TG levels and BMI. The results demonstrated that the expression levels of HMGB1 and TLR4 in patients with T2DM or obesity were increased, which were associated with glycolipid metabolism disorders. Therefore, the HMGB1/TLR4 may serve a role in inflammatory process associated with obesity and T2DM.

Latroeggtoxin-VI (LETX-VI) is a peptide neurotoxin discovered from Latrodectus tredecimguttatus eggs. In the current study, the action features of the neurotoxin on PC12 cells were systematically investigated. LETX-VI could promote dopamine release from PC12 cells in the absence and presence of Ca2+, involving an even more complex action mechanism in the presence of Ca2+ and when the treatment time was longer. Although LETX-VI enchanced the autophagy and secretion activity in PC 12 cells, it showed no remarkable influence on the proliferation, cell cycle, apoptosis and ultrastructure of the cells. Pulldown combined with CapLC-MS/MS analysis suggested that LETX-VI affected PC12 cells by interacting with multiple proteins involved in the metabolism, transport, and release of neurotransmitters, particularly dopamine. The low cytotoxicity and effective regulatory action of LETX-VI on PC12 cells suggest the potential of the active peptide in the development of drugs for the treatment of some dopamine-related psychotic diseases and cancers.

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.间斑寇蛛Latrodectus tredecimguttatus的一个显著特点是其毒腺外组织甚至卵粒中也存在毒性成分。研究毒腺外的毒素不但可以加深对蜘蛛毒素的了解，而且可以发现具有重要应用前景的新型毒素分子。为了探究间斑寇蛛卵粒中低丰度表达的蛋白质类毒素，利用生物信息学方法从间斑寇蛛卵粒转录组中挖掘出一条编码多肽毒素的基因序列，利用基于3′-RACE和巢式PCR的策略成功克隆并异源表达了该基因。表达的多肽毒素命名为间斑寇蛛卵粒毒素-Ⅵ (Latroeggtoxin-Ⅵ，LETX-Ⅵ)。生物学活性鉴定结果表明，LETX-Ⅵ能抑制ND7/23细胞膜上的钠离子通道电流和促进PC12细胞多巴胺的释放，但对美洲蜚蠊Periplaneta americana和金黄色葡萄球菌及白色念珠菌等细菌和真菌不显示明显的毒性，说明LETX-Ⅵ是一种哺乳动物特异的神经毒素，在神经生物学研究工具试剂和相关疾病治疗药物的研发等方面具有潜在的应用前景。.

Latroeggtoxin-VI (LETX-VI) is a peptide neurotoxin newly found from the eggs of spider L. tredecimguttatus. To explore the mechanism of action of the LETX-VI on nerve cells, the effects of LETX-VI on PC12 cells, a commonly used neuron model, were analyzed using a pull-down assay-guided strategy. LETX-VI was shown to interact with 164 PC12 cell proteins that have diverse molecular functions such as binding, catalysis, regulation, structural activity, etc., thereby extensively affecting the biological processes in the PC12 cells, particularly protein metabolism, response to stimulus, substance transport, and nucleic acid metabolism, with 56.71%, 42.07%, 29.88% and 28.66% of the identified proteins being involved in these biological processes, respectively. By interacting with the relevant proteins, LETX-VI enhanced the synthesis of dopamine, positively regulated cell division and proliferation, and negatively regulated cell cycle arrest, cell death, and apoptotic processes, and therefore has limited cytotoxicity against the PC12 cells, which were further experimentally confirmed. In general, the effects of LETX-VI on PC12 cells are more regulatory than cytotoxic. These findings have deepened our understanding of the action mechanism of LETX-VI on nerve cells and provided valuable clues for further related researches including those on Parkinson’s disease.

Latrodectus species are among the most venomous of spiders, with abundant toxic proteinaceous components in their venomous glands and other tissues, as well as their eggs. To date, several proteinaceous toxins with insecticidal potential, including α-insectotoxin and δ-insectotoxin, two of the most potent known insecticidal toxins, have been purified and characterized by comprehensively utilizing conventional biochemical techniques. This has greatly enhanced our knowledge of the molecular basis and mechanism of action of their toxicity. Application of proteomic and transcriptomic techniques further revealed the synergistic action of multiple Latrodectus proteinaceous toxins and toxin-like components. Insecticidal toxins from Latrodectus spiders have great potential in insect pest control; however, more studies are needed to further reveal their mechanisms of action and understand their structures and properties before any practical application, for example, the insecticidal toxin-containing fusion proteins with oral activity. Here, we review current knowledge of the molecular basis and mechanism of action underlying the insecticidal activity of venoms and toxins from Latrodectus spiders, and examine their potential application in insect pest control. © 2018 Society of Chemical Industry.

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca2+-independent and Ca2+-dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca2+-dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca2+-dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca2+-dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca2+-dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 9999: 1–10, 2017. © 2017 Wiley Periodicals, Inc.

Black widow spiders contain toxic components not only in the venom glands but also in other parts of the spider body, including the legs and abdomen. Additionally, both the eggs and newborn spiderlings of the black widow spider contain venom. It is important to investigate their potential effects on cancer cells. In the present study, the effects of newborn black widow spiderling extract on human HeLa cells were evaluated in vitro. When applied at different concentrations, the total extract decreased HeLa cell viability in a dose-dependent manner, with an IC50 value of 158 µg/ml. Flow cytometry indicated that treatment of HeLa cells with the total extract of the spiderlings induced apoptosis in HeLa cells in a dose-dependent manner and led to cell cycle arrest in the S-phase. Additionally, application of the total extract at different concentrations increased apoptosis-related caspase 3 activity in a dose-dependent manner. HeLa cells treated with the total extract appeared to be morphologically changed, exhibiting membrane blebbing, nuclear fragmentation and condensation of chromatin. Further separation and activity screening demonstrated that the cytotoxic and apoptotic activities of the total extract were attributable mainly to its high molecular mass proteins, one of which was purified and characterized to determine its anti-tumor activities on HeLa cells. The results of the present study therefore have expanded understanding regarding the effect of spider toxins on cancer cells and suggested that components of black widow spiderlings may be developed as a promising novel agent to treat cancer.

Synaptotagmin I (Syt I) functions in the regulation of neurotransmitter release and multiple other cellular processes through its C2 domain binding to other molecules. Our previous study demonstrated that Rab3A, a small GTP-binding protein, is a new interacting partner of Syt I and could bind to both of the C2 domains; the polylysine motif in C2B is a key site for Rab3A binding, but the binding site on C2A is not clear. In order to localize Rab3-binding site on C2A and reveal the relevant regulatory mechanism, in the present study we investigated the interaction between recombinant Rab3A and various C2A mutants. The results showed that a key Rab3A-binding site on C2A is located at R199K200 in the flexible loop 2 of the domain, and the site does not overlap with most of the known functional sites or residues. It was speculated that the interaction between Rab3A and C2A is not simply based on electrostatic force, and Rab3A regulates C2A-mediated vesicle-presynaptic membrane fusion mainly through affecting the C2A binding to phospholipids in the presynaptic membrane. These results have contributed to the comprehension of action mechanism of Rab3 and synaptotagmin in the regulation of synaptic vesicle exocytosis.

The early reports and our previous work confirmed the existence of the toxic proteinaceous components in the body of the L. tredecimguttatus newborn and adult spiders. For revealing the differences in the protein expression profiles and toxicity bases of the spiders at different developmental stages, the spiderling and adult spider proteins were comparatively analyzed using a proteomic strategy. Totals of 429 and 958 proteins were identified from the spiderlings and adult spiders, respectively, with 239 proteins being identified from both of them. Although some similarities between the spiderling and adult spider proteomes exist, there are obvious differences between the two proteomes in size, complexity, molecular weight (MW) distribution, acid-base property, and hydropathicity, etc. Gene ontology (GO) analysis demonstrates that, comparing based on the percentages of proteins, the spiderling and adult spider proteins have generally similar distribution profiles with respect to the subcellular localization, molecular function and biological process. However, there are still some differences between these two sets of proteins in some classifications of the three GO categories. For the adult spiders, latrotoxins together with other toxins and toxin-like proteins, etc. constitute their toxicity basis, whereas the toxicity of the spiderlings depends mainly on the synergistic action of atypical latrotoxins and toxin-like proteins, most of which are different from those of the adult spiders, demonstrating that the spiders at different developmental stages have largely different toxicity mechanisms.

Membrane proteins solubilized in a starting buffer containing high concentration of SDS are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion, and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins, especially integral membrane proteins.

Latrodectus species are among the most venomous of spiders, with abundant toxic proteinaceous components in their venomous glands and other tissues, as well as their eggs. To date, several proteinaceous toxins with insecticidal potential, including α-insectotoxin and δ-insectotoxin, two of the most potent known insecticidal toxins, have been purified and characterized by comprehensively utilizing conventional biochemical techniques. This has greatly enhanced our knowledge of the molecular basis and mechanism of action of their toxicity. Application of proteomic and transcriptomic techniques further revealed the synergistic action of multiple Latrodectus proteinaceous toxins and toxin-like components. Insecticidal toxins from Latrodectus spiders have great potential in insect pest control; however, more studies are needed to further reveal their mechanisms of action and understand their structures and properties before any practical application, for example, the insecticidal toxin-containing fusion proteins with oral activity. Here, we review current knowledge of the molecular basis and mechanism of action underlying the insecticidal activity of venoms and toxins from Latrodectus spiders, and examine their potential application in insect pest control. © 2018 Society of Chemical Industry.

Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins. By functional enrichment analysis, 287 molecular function terms for the PM proteome and 173 for the MM proteome were obtained. The MM proteome had a lower percentage of binding function terms and a higher percentage of catalysis function terms than the PM proteome, suggesting that mitochondrial proteins were more inclined to affect the physiological and biochemical processes by binding various molecules and as enzymes. Biological process enrichment showed that the genes of the PM and MM proteomes were mapped to 1104 and 460 biological processes, respectively. The biological processes with the most mapped genes of the PM proteome included those involved in vesicle recycling, transmitter release, neuronal development, protein and ion transport, etc., whereas those involved in electron transport, ATP synthesis, mitochondrial transport, mitochondrial apoptosis, etc., were the most gene-mapped biological processes for the MM proteome. The present work has deepened our understanding of the structure and function of hippocampal neurons and provided reference methods for research in the related field. Graphical abstract Functional comparison of the plasma membrane and mitochondrial membrane proteomes.

To enhance light extraction efficiency, high-quality InGaN/GaN multiple quantum well (MQW) LED was grown on cone-shaped patterned sapphire substrate (CPSS) by using metal organic chemical vapor deposition (MOCVD). In the growth of InGaN multiple quantum well structure, all V-defects are not always connected with threading dislocations (TDs) at their bottom, also many V-defects are generated from the stacking mismatch boundaries induced by stacking faults which are formed within the MQW due to the strain relaxation. The formation of the V-defect is kinetically controlled by reduced Ga incorporation on the pyramid walls ( { 10 1 ¯ 1 } planes). The improvement of luminous intensity and reverse I–V characteristic (the leakage currents of the LEDs grown on CPSS and USS were − 0.23 μ A and − 0.76 μ A , respectively, at the reverse voltage of −10 V) was explained by considering not only an increase of the extraction efficiency via the suppressed total internal reflection at the CPSS interface but also a decrease of V-defect density.

Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.

The relative quantification and identification of proteins by matrix-assisted laser desorption ionization time-of-flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N-terminal derivatization with 4-sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while 18O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4-sulphophenyl isothiocyanate derivatization with 18O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R2 value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD50 being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage-activated Na(+) , K(+) , and Ca(2+) channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity. Language: en

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

It has been reported that Heteropodatoxin3 (HpTx3), a peptidic neurotoxin purified from the venom of the spider species Heteropoda venatoria, could inhibit Kv4.2 channels. Our present study newly found that HpTx3 also has potent and selective inhibitory action on Nav1.7, with an IC50 of 135.61 &plusmn, 12.98 nM. Without effect on the current&ndash, voltage (I-V) relationship of Nav1.7, HpTx3 made minor alternation in the voltage-dependence of activation and steady-state inactivation of Nav1.7 (4.15 mV and 7.29 mV, respectively) by interacting with the extracellular S3&ndash, S4 loop (S3b&ndash, S4 sequence) in domain II and the domain IV of the Nav channel subtype, showing the characteristics of both pore blocker and gate modifier toxin. During the interaction of HpTx3 with the S3b&ndash, S4 sequence of Nav1.7, the amino acid residue D in the sequence played a key role. When administered intraperitoneally or intramuscularly, HpTx3 displayed potent analgesic activity in a dose-dependent manner in different mouse pain models induced by formalin, acetic acid, complete Freund&rsquo, s adjuvant, hot plate, or spared nerve injury, demonstrating that acute, inflammatory, and neuropathic pains were all effectively inhibited by the toxin. In most cases HpTx3 at doses of &ge, 1mg/kg could produce the analgesic effect comparable to that of 1 mg/kg morphine. These results suggest that HpTx3 not only can be used as a molecular probe to investigate ion channel function and pain mechanism, but also has potential in the development of the drugs that treat the Nav1.7 channel-related pain.

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S—. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:337-342, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21493

The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.

Previous work found that high-molecular-weight fractions in the egg extract of Latrodectus tredecimguttatus exhibited strong toxicities. For investigating the possible relationship of proteins in the eggs with the toxic effect, the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spider's venom. SDS-PAGE showed that the proteins of eggs were primarily distributed in the molecular weight range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular weights of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract, which were involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are few similarities between the protein composition of the two materials, demonstrating that the eggs have their own distinct toxic mechanism.

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca

[EN] Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals., We thank Dr. Alice Cheung, Dr. Daniel Moura, Grossniklaus Ueli, Dr. Jigang Li, and Dr. Nieng Yan for providing plant, ABI1 antibody, or plasmid materials, and Dr. Legong Li for assistance in laser confocal microscopy. This work was supported by grants from National Natural Science Foundation of China (NSFC-31400232, 31571444), the State Key Laboratory of Molecular Developmental Biology (2015-MDB-KF-12), the Fundamental Research Funds for the Central Universities of China, and a grant from the National Science Foundation.

Background Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear. Results In this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared. Conclusions The present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies.

Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.