AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two Orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, Oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three Pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

A unified view on enzyme catalysis by cryo-EM study of a DNA topoisomerase.

The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the prevalence and consensus of these theories require further examination. Here we use cryogenic electron microscopy and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate substrate binding theories in a joint and ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, then induced fit. The apo-AsfvTop2 pre-exists in six conformers that comply with the two-gate mechanism directing DNA passage and release in the Top2 catalytic cycle. The structures of AsfvTop2-DNA-inhibitor complexes show that substantial induced-fit changes occur locally from the closed apo-conformer that however is too far-fetched for the open apo-conformer. Furthermore, the ATPase domain of AsfvTop2 in the MgAMP-PNP-bound crystal structures coexist in reduced and oxidized forms involving a disulfide bond, which can regulate the AsfvTop2 function.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

A class I TGA transcription factor from Tripterygium wilfordii Hook.f. modulates the biosynthesis of secondary metabolites in both native and heterologous hosts.

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.

Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation.

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Knockdown of PEBP4 suppresses proliferation, migration and invasion of human breast cancer cells.

Phosphatidylethanolamine-binding protein 4 (PEBP4), a member of the PEBP family, plays a pivotal role in tumor progression. However, the roles of PEBP4 in breast cancer remain unclear. Therefore, in the present study, we investigated the effects of PEBP4 on breast cancer cell proliferation, migration and invasion, and the underlying mechanism was also explored. Our results showed that the expression of PEBP4 was significantly up-regulated in breast cancer cell lines. Knockdown of PEBP4 inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. Furthermore, knockdown of PEBP4 suppressed breast cancer cell migration and invasion with prevented EMT. Mechanistically, knockdown of PEBP4 inhibited breast cancer cell proliferation and migration through the inactivation of PI3K/Akt signaling pathway. In conclusion, the present study demonstrated for the first time that knockdown of PEBP4 inhibited the proliferation, invasion and tumorigenesis in breast cancer cells. Thus, PEBP4 may serve as a potential therapeutic target for the treatment of breast cancer.

HPIP promotes thyroid cancer cell growth, migration and EMT through activating PI3K/AKT signaling pathway.

Hematopoietic pre-B cell leukemia transcription factor (PBX)-interacting protein (HPIP), a co-repressor for the transcription factor PBX, is a nucleo-cytoplasmic shuttling protein. Increasing evidence suggests that HPIP is an oncogene which is frequently overexpressed in many human carcinomas. However, the role of HPIP in thyroid carcinoma is still unclear. Therefore, in this study, we investigated the role of HPIP in thyroid carcinoma, and explored the underling mechanism. We found that the expression of HPIP is upregulated in thyroid carcinoma cell lines. Knockdown of HPIP inhibits thyroid carcinoma cell proliferation, migration/invasion and epithelial-mesenchymal transition (EMT). HPIP knockdown also reduces thyroid tumor growth in nude mice. Furthermore, knockdown of HPIP significantly inhibits the expression of phosphorylated PI3K and AKT in thyroid carcinoma cells. Taken together, these results suggest that knockdown of HPIP inhibits the proliferation, migration and EMT by suppressing the PI3K/AKT pathway, and HPIP may be a potential therapeutic target for the treatment of thyroid carcinoma.

Functional regulation of Zfp36l1 and Zfp36l2 in response to lipopolysaccharide in mouse RAW264.7 macrophages.

BACKGROUND: The tristetraprolin (TTP) family of mRNA-binding proteins contains three major members, Ttp, Zfp36l1, and Zfp36l2. Ttp down-regulates the stability of AU-rich element-containing mRNAs and functions as an anti-inflammation regulator. METHODS: To examine whether other TTP family proteins also play roles in the inflammatory response, their expression profiles and the possible mRNA targets were determined in the knockdown cells. RESULTS: Ttp mRNA and protein were highly induced by lipopolysaccharide (LPS), whereas Zfp36l1 and Zfp36l2 mRNAs were down-regulated and their proteins were phosphorylated during early lipopolysaccharide stimulation. Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp. Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life. Increasing the expression of Mkp-1 inhibited the activation of p38 MAPK under lipopolysaccharide stimulation and down-regulated Tnfα, and Ttp mRNA. In addition, hyper-phosphorylation of Zfp36l1 might stabilize Mkp-1 expression by forming a complex with the adapter protein 14-3-3 and decreasing the interaction with deadenylase Caf1a. CONCLUSIONS: Our findings imply that the expression and phosphorylation of Zfp36l1 and Zfp36l2 may modulate the basal level of Mkp-1 mRNA to control p38 MAPK activity during lipopolysaccharide stimulation, which would affect the inflammatory mediators production. Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.

The RNA recognition motif of NIFK is required for rRNA maturation during cell cycle progression.

Ribosome biogenesis governs protein synthesis. NIFK is transactivated by c-Myc, the key regulator of ribosome biogenesis. The biological function of human NIFK is not well established, except that it has been shown to interact with Ki67 and NPM1. Here we report that NIFK is required for cell cycle progression and participates in the ribosome biogenesis via its RNA recognition motif (RRM). We show that silencing of NIFK inhibits cell proliferation through a reversible p53-dependent G1 arrest, possibly by induction of the RPL5/RPL11-mediated nucleolar stress. Mechanistically it is the consequence of impaired maturation of 28S and 5.8S rRNA resulting from inefficient cleavage of internal transcribed spacer (ITS) 1, a critical step in the separation of pre-ribosome to small and large subunits. Complementation of NIFK silencing by mutants shows that RNA-binding ability of RRM is essential for the pre-rRNA processing and G1 progression. More specifically, we validate that the RRM of NIFK preferentially binds to the 5'-region of ITS2 rRNA likely in both sequence specific and secondary structure dependent manners. Our results show how NIFK is involved in cell cycle progression through RRM-dependent pre-rRNA maturation, which could enhance our understanding of the function of NIFK in cell proliferation, and potentially also cancer and ribosomopathies.

[Analysis and assessment of atmospheric pollution based on accumulation characterization of heavy metals in Platanus acerifolia leaves].

The present work was aimed to evaluate the heavy metal pollution in the atmosphere of Huainan City. We measured and clustered the accumulation of six heavy metals in Platanus acerifolia leaves in 20 sampling fields with six types of environmental conditions, and analyzed the EF value of heavy metal enrichment in the leaves. The results showed that the accumulations in Platanus acerifolia leaves varied according to different types of metals, following the order of Zn > Cu > Cr > Ni > Pb > Cd. Environmental conditions also had great influence on the accumulation of heavy metals. Cd and Cu were mostly found in cement plant and mine, respectively, and Cr, Ni, Pb and Zn were significant higher in main road, compared with other environmental conditions. The average values of EF for all the metals expect Cr in scenic and village area were over 1. The average values of EF for all the metals in mine, power plant, main road and cement plant were above 3. The overall pollution condition of heavy metals in Huainan City followed the order of Cd > Cu > Zn > Ni > Pb > Cr.

Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo.

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.

[Leaf micro-morphology and features in adsorbing air suspended particulate matter and accumulating heavy metals in seven trees species].

The purpose of this study was to assess the relationship between tree leaf micro-morphology and features in adsorbing air suspended particulate matter and accumulating heavy metals. Seven tree species, including Ginkgo biloba, at heavy traffic density site in Huainan were selected to analyze the frequency of air particulate matter retained by leaves, the particle amount of different sizes per unit leaf area retained by leaves and its related micro-morphology structure, and the relationship between particle amount of different sizes per unit leaf area retained by leaves and its related accumulation of heavy metals. We found that the species characterized by small leaf area, special epidemis with abundant fax, and highly uneven cell wall, as well as big and dense stomata and without trichomes mainly absorbed fine particulate matter; while those species with many trichomes mainly retained coarse particulate matter. Accumulation of heavy metals in leaves of the seven species was significantly different except for Ph. Tree species with high capacities in heavy metal accumulation were Ginkgo biloba, Ligustrum lucidum, and Cinnamomum camphora. Accumulation of Cd, Cr, Ni, Zn, Cu and total heavy metal concentration for seven tree species was positively related to the amount of particulate matter absorbed. Correlation coefficients of d10 vs d2.5, d10 vs d1.0, d2.5 vs d1.0 were 0.987, 0.971, 0.996, respective, and the correlate level was significant. The ratios of d2.5/d10, d1.0/d10, d1.0/d2.5 were 0.844, 0.763, 0.822, indicating that the particulate matter from traffic was mainly fine particulates.

Phosphorylation of mRNA decapping protein Dcp1a by the ERK signaling pathway during early differentiation of 3T3-L1 preadipocytes.

BACKGROUND: Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5' cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway-mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.

Tristetraprolin inhibits poly(A)-tail synthesis in nuclear mRNA that contains AU-rich elements by interacting with poly(A)-binding protein nuclear 1.

BACKGROUND: Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS: To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element-containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain-deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element-containing tumor necrosis factor α/luciferase mRNA construct. CONCLUSION/SIGNIFICANCE: In addition to its known cytosolic mRNA-degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element-containing mRNA.

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes.

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.

Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-κB activation.

The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.

Drosophila eyes absent is a novel mRNA target of the tristetraprolin (TTP) protein DTIS11.

The Tristetraprolin (TTP) protein family includes four mammalian members (TTP, TIS11b, TIS11d, and ZFP36L3), but only one in Drosophila melanogaster (DTIS11). These proteins bind target mRNAs with AU-rich elements (AREs) via two C3H zinc finger domains and destabilize the mRNAs. We found that overexpression of mouse TIS11b or DTIS11 in the Drosophila retina dramatically reduced eye size, similar to the phenotype of eyes absent (eya) mutants. The eya transcript is one of many ARE-containing mRNAs in Drosophila. We showed that TIS11b reduced levels of eya mRNA in vivo. In addition, overexpression of Eya rescued the TIS11b overexpression phenotype. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3' UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila S2 cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability. Our results suggest that eya mRNA is a target of TTP proteins, and that downregulation of EYA by TTP may lead to reduced cell viability in Drosophila and human cells.

Essential roles of p53 and MAPK cascades in microcystin-LR-induced germline apoptosis in Caenorhabditis elegans.

Hepatotoxin microcystin-LR (MC-LR) can induce apoptosis in a variety of cells. However, the underlying pathways of MC-LR-induced apoptosis have not been well elucidated yet. To find out the roles of underlying pathways in apoptosis signaling in response to MC-LR, germ cell corpses were scored in Caenorhabditis elegans N2 wild type and strains carrying mutated alleles homologous to their mammalian counterparts. We found that exposure to MC-LR at 1.0 µg/L significantly increased germline apoptosis in N2. Germline apoptosis was absent at all doses in ced-3 and ced-4 loss-of-function strains. MC-LR-induced apoptosis was blocked in Bcl-2 gain-of-function strain ced-9(n1950), whereas it showed a slight increase in BH3-only protein EGL-1 mutated strain. The null mutation of cep-1, which is the homologue of p53 tumor suppressor gene, significantly inhibited MC-LR-induced cell death, and checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects. Apoptosis in loss-of-function members of ERK, JNK, and p38 MAPK signaling pathways reduced significantly under MC-LR exposure, and members of MAPKK subgroup JKK-1, MEK-1, and SEK-1 worked cooperatively. Our results show that the caspase protein CED-3 and Apaf-1 protein CED-4 were absolutely required for the apoptotic processes, and that the p53/CEP-1 and MAPKs cascades played essential roles in modulating MC-LR-induced germline apoptosis in C. elegans.

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam.

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 x 10(-6) at dose of 31.2 x 10(14) ions/cm2, which was about 18-fold over the background (1.5 x 10(-6)) and 10-fold over the vacuum controls (2.6 x 10(-6)). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or -TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or -TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level +/-TGGC and high proportion additions than those of gamma-radiation induced, implying there were some different effects or processes between them.

Carp ovarian cystatin binds and agglutinates spermatozoa via electrostatic interaction.

A sperm-agglutinating factor was purified from ovulated carp eggs and the conditioned medium (CM) of cortical-reacted eggs. It was identified to be the carp ovarian cystatin. Three cystatin isoforms were found. The cystatin isolated from the CM had a higher sperm-agglutinating activity than that isolated from eggs, although the cystatins have identical N-terminal amino acid sequences, masses, and positive charges. Differences in sperm-agglutinating activity between the cystatins of the CM and eggs may be caused by the different conformations because they differed in circular dichroism spectrum and tryptic map. Cystatin was discharged from cortical granules to the perivitelline space after fertilization and is abundant in the perivitelline fluid (PVF) of early stage embryos. Cystatin rapidly agglutinated spermatozoa via an electrostatic interaction. Other basic proteins also agglutinated carp spermatozoa. Their activities were inhibited by salt and high pH. Cystatin bound to the entire surface of carp spermatozoa. The PVF of early embryos agglutinated carp spermatozoa. The activity was related to the cystatin content and influenced by ionic strength and pH. Therefore, cystatin is the major sperm-agglutinating factor of PVF. Owing to the rapid action of cystatin on spermatozoa agglutination and the presence of a high concentration of cystatin in PVF, cystatin is considered important for preventing polyspermy in carp eggs.