Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca2+, Mg2+, Mn2+, and Fe2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases.IMPORTANCE Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production, thereby maintaining the efficiency of the megasynthase involved in this class of secondary metabolites' biosynthesis. Specifically, this class of enzyme is believed to be involved in editing misprimed precursors, controlling initial units, providing key intermediates, and releasing final synthetic products in the biosynthesis of this class of secondary metabolites. Our results indicate that the putative TEII CalG present in the calcimycin (A23187)-producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or for the release of the product compound from, the megasynthase involved in calcimycin biosynthesis. Rather, the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolites.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 µM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

HigA2 (Rv2021c) Is a Transcriptional Regulator with Multiple Regulatory Targets in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 (Rv2022c), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb.

The Restriction Activity Investigation of Rv2528c, an Mrr-like Modification-Dependent Restriction Endonuclease from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), as a typical intracellular pathogen, possesses several putative restriction-modification (R-M) systems, which restrict exogenous DNA's entry, such as bacterial phage infection. Here, we investigate Rv2528c, a putative Mrr-like type IV restriction endonuclease (REase) from Mtb H37Rv, which is predicted to degrade methylated DNA that contains m6A, m5C, etc. Rv2528c shows significant cytotoxicity after being expressed in Escherichia coli BL21(DE3)pLysS strain. The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay indicates that Rv2528c cleaves genomic DNA in vivo. The plasmid transformation efficiency of BL21(DE3)pLysS strain harboring Rv2528c gene was obviously decreased after plasmids were in vitro methylated by commercial DNA methyltransferases such as M.EcoGII, M.HhaI, etc. These results are consistent with the characteristics of type IV REases. The in vitro DNA cleavage condition and the consensus cleavage/recognition site of Rv2528c still remain unclear, similar to that of most Mrr-family proteins. The possible reasons mentioned above and the potential role of Rv2528c for Mtb were discussed.

Mutasynthesis of pyrrole spiroketal compound using calcimycin 3-hydroxy anthranilic acid biosynthetic mutant.

The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.

C-N bond formation by a polyketide synthase.

Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique "∞"-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C-C bond formation can be tweaked to mediate C-N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.

A novel single-chain-Fv antibody against connective tissue growth factor attenuates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) has been identified as playing critical roles in fibrosis and is a promising therapeutic target. In a previous study, we used a phage display library to develop a humanized single-chain variable fragment antibody (scFv) against CTGF. In the present study, the protective effect of anti-CTGF scFv against bleomycin (BL)-induced pulmonary fibrosis was investigated in mice. METHODS: The expression of α-smooth muscle actin in human embryonic lung fibroblast (HELF) cells was analysed by western blotting. A mouse model of pulmonary fibrosis was established by tracheal injection of BL (5 mg/kg). Mice received anti-CTGF scFv (4 mg/kg, three times a week) by i.v. injection. The effects of anti-CTGF scFv were evaluated by leukocyte counts in BAL fluid, hydroxyproline measurements in lung tissue and pathological examination. RESULTS: α-Smooth muscle actin expression was decreased in HELF cells treated with anti-CTGF scFv. Anti-CTGF scFv significantly reduced the numbers of inflammatory leukocytes (total and differential count) in BAL fluid, as well as the hydroxyproline content of lung tissue. The severity of alveolitis and fibrosis in the mouse model was markedly attenuated by treatment with anti-CTGF scFv. CONCLUSIONS: Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis.

Bioscreening of phage display antibody library and expression of a humanized single-chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2).

Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.

A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (ΔcalR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in calcimycin biosynthesis and suggest that calcimycin production can be improved by manipulating its biosynthetic machinery.

G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-κB and cell proliferation.

G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi­quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)­N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'­diamidino­2­phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-κB reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over­expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation.

Analysis of AC3-33 gene expression in multiple organ cancer and adjacent normal tissue by RNA in situ hybridization.

The AC3-33 gene encodes a secretory protein that can inhibit Elk1 transcriptional activity via the ERK1/2 pathway. In the current study, in situ RNA hybridization was used to detect the AC3-33 gene expression in multiple organ cancer and cancer-adjacent normal tissue. The results showed that the expression level of AC3-33 varies across different tissues. AC3-33 exhibited positive expression in squamous cell carcinoma of the esophagus, adenocarcinoma of the rectum, hepatocellular carcinoma, squamous cell carcinoma (SCC) of the lung, cancer-adjacent normal hepatic tissue, clear cell carcinoma of the kidney, invasive ductal carcinoma of the breast, SCC of the uterine cervix and cancer-adjacent normal kidney tissue. Negative expression of AC3-33 was observed in adenocarcinoma of the stomach and colon, cancer-adjacent normal esophageal tissue, cancer-adjacent normal gastric tissue, cancer-adjacent normal colon tissue, cancer-adjacent normal rectal tissue, serous adenocarcinoma of the ovary and cancer-adjacent normal ovarian tissue. However, the expression of AC3-33 in cancer adjacent normal breast tissue was partially positive. In conclusion, the AC3-33 gene does exhibit positive expression in certain carcinomas, which may indicate that AC3-33 has a significant involvement in the development and progression of these carcinomas.

MicroRNA profiles and potential regulatory pattern during the early stage of spermatogenesis in mice.

Spermatogenesis is a complicated and poorly understood process that relies on the precise regulation of the self-renewal and differentiation of spermatogonia. In many organisms, microRNAs (miRNAs) are involved in multiple developmental processes as critical regulators of transcriptional and post-transcriptional gene silencing. This study investigated the expression pattern of miRNAs in type B spermatogonia cells (BSc) and primary spermatocytes (PSc) of mice, using a high-throughput small RNA sequencing system. The results revealed that the expression levels of Let-7 family miRNAs were remarkably high in both cell types. Furthermore, the expression levels of miR-21, miR-140-3p, miR-103, miR-30a, miR-101b and miR-99b were decreased during the transformation from BSc to PSc. These miRNAs target vital genes that participate in apoptosis, cell proliferation and differentiation, junction assembly and cell cycle regulation. These results highlight the indispensable role of miRNAs in spermatogenesis.

Analysis of TMEM174 gene expression in various renal cancer types by RNA in situ hybridization.

Transmembrane protein 174 (TMEM174) mRNA is easily detectable in human kidney tissues and activates AP-1 and promotes 293T cell proliferation. In the present study, RNA in situ hybridization was used to detect TMEM174 gene expression in various malignant renal cancer and normal renal tissues. The results showed that TMEM174 exhibits differential expression in renal tissues, with a high positive rate of expression in squamous cell carcinoma with necrosis, papillary renal cell carcinoma and transitional cell carcinoma, and a low positive rate of expression in clear cell carcinoma, interstitial nephritis, undifferentiated carcinoma, retroperitoneal metastatic clear cell carcinoma, adrenal gland metastatic clear cell carcinoma, pelvic cavity metastatic chromophobe carcinoma, severe atypical hyperplasia of transitional epithelium and hyperplasia. Extremely weak expression was exhibited in collecting duct carcinoma, Wilms' tumor, chronic pyelonephritis, acute pyelonephritis, cancer adjacent normal renal tissue and normal renal tissue. In conclusion, the TMEM174 gene exhibited high expression levels in certain renal carcinomas, which may indicate that TMEM174 may have a significant role in the development and progression of these renal carcinomas.

δEF1 upregulates CDK4 transcription via the E2-box element on the CDK4 promoter.

The zinc finger-homeodomain transcription factor, δ-crystallin enhancer factor 1 (δEF1) has been identified as a regulatory factor involved in the promotion of breast cancer cell proliferation via the downregulation of p21 and the upregulation of cyclin-dependent kinase-2 (CDK2) and CDK4 expression. However, the molecular mechanisms underlying the regulation of CDK4 expression by δEF1 have not yet been elucidated. The present study demonstrated that the ectopic expression of δEF1 in MDA-MB-231 breast cancer cells significantly increased the activity of the CDK4 promoter. Deletion of the E2-box (CACGTG), which is located at position -197/-191 on the human CDK4 promoter, significantly attenuated the activation of CDK4 transcription by δEF1. In addition, a CDK4 promoter-M construct was generated via site-directed mutagenesis of the E2-box on the human CDK4 promoter. Luciferase assay showed that the activation of CDK4 promoter-M activity by δEF1 was markedly decreased compared with the CDK4-promoter-0.4k promoter. Knockdown of δEF1 using RNA interference resulted in the inhibition of CDK4 promoter activity. These observations suggest that δEF1 upregulates CDK4 transcription via the E2-box element on the CDK4 promoter.

Analysis of promoters and CREB/AP-1 binding sites of the human TMEM174 gene.

Transmembrane protein 174 (TMEM174) is a type III transmembrane protein with no clear signal peptide. The N and C terminals are located inside the cell. Our previous study demonstrated high expression of TMEM174 in the kidney and its potential involvement in renal cancer based on its capacity to stimulate cell proliferation. However, the mechanism by which TMEM174 promotes proliferation at the transcriptional level remains to be elucidated. In the present study, the TMEM174 promoter region was amplified from whole blood DNA. Six different regions of the regulatory sequences of the TMEM174 promoter region including ~2.5 kb of the upstream region were cloned into the dual luciferase expression vector pGL3-basic. Comparison of the activity of these fragments in dual luciferase reporter assays revealed higher levels of activity for the fragments spanning -186 to +674, -700 to +674, -1,000 to +674 and -2,500 to +1 bp. Lower levels of activity were detected for the fragments spanning -466 to +674 and -890 to +674 bp. The highest activity was detected for the fragment spanning -186 to +674 bp. Electrophoretic mobility shift assay (EMSA) was performed to determine effective transcription factor binding sites. Specific binding of the cyclic-AMP response element binding (CREB) within the TMEM174 gene promoter region was demonstrated, although binding of the activator protein-1 (AP-1) was also detected in this region. In conclusion, these results suggest that the core promoter region of the human TMEM174 gene is located within the region spanning -186 to +674 bp and that the transcription factors CREB and AP-1 are involved in the transcriptional regulation of this gene.

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882.

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 µM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.

Role of LM23 in cell proliferation and apoptosis and its expression during the testis development.

LM23, a gene expressed specifically in the testis in a stage-specific manner, has a diverse range of functions that are important in both the life and death of spermatogenic cells. The aim of this study was to further investigate the expression of LM23 in the developing rat testis and the biological function of LM23 in proliferation and antiapoptosis in vitro. Semiquantitative reverse transcription (RT)-PCR and real-time PCR were used to examine the expression of LM23 in testis at different developmental stages. The results suggested that LM23 mRNA levels in the testis increased progressively after birth. The role of LM23 in proliferation was analyzed with cell counting kit-8 (CCK8), colony-forming efficiency (CFE) and flow cytometry assays. The results indicated that ectopic expression of LM23 in 293T cells significantly promoted cell proliferation by increasing cell numbers in S phase. Several methods were used, including CCK8, annexin V and propidium iodide staining and western blotting, to determine the role of LM23 in apoptosis. The results showed that LM23 played a protective role in H2O2-induced apoptosis of 293T cells, mediated at least in part through the Akt/PI3K signal pathway. Taken together, these results provide new insights into the role of LM23 in the development of the testes and spermatogenesis.

[Effects of climate factors on the epidemic of apple Marssonina blotch in Shaanxi Province and related prediction models].

Based on the long term (1999-2008) monitoring of air temperature and relative humidity and of the occurrence and epidemiological trend of Marssonina blotch in the main apple-production area of Shaanxi Province, this paper analyzed the occurrence time, pathogenesis regularity, and epidemiological level of Marssonina blotch, with the 1- and 3-dimensional models for predicting Marssonina blotch under effects of ten-day mean air temperature (T) and relative humidity (Hm) constructed. In study area, the development of Marssonina blotch was mainly affected by environment factors. This disease spread rapidly in field in July and August, causing orchard defoliation, and the harm persisted until September. After the first frost, new disease spots no longer developed. The data of T and Hm in the models showed a good fitting with field condition. The 3-dimensional dynamic prediction model of Marssonina blotch was f(T, Hm) = -0.0172 T3 + 0.9497T2--16.2209T + 88.9923-0.00001Hm3 + 0.00354Hm2--0.15554Hm + 2.36578, where f(T, Hm) was disease index. The modeling results showed that the T for the occurrence of Marssonina blotch in field was 15 degrees C, and the disease would have an epidemic peak when the T and Hm in July and August reached 23 degrees C and > or = 90%, respectively.

Interaction of antimicrobial peptide s-thanatin with lipopolysaccharide in vitro and in an experimental mouse model of septic shock caused by a multidrug-resistant clinical isolate of Escherichia coli.

s-thanatin, an analogue of thanatin, was synthesised by substituting the fifteenth amino acid threonine with serine and showed broad antimicrobial activity against Gram-negative and Gram-positive bacteria. To evaluate its antimicrobial activity against a multidrug-resistant (MDR) clinical isolate as well as its anti-endotoxin activity, its lipopolysaccharide (LPS)-binding and -neutralising activity in vitro and its therapeutic efficacy in an experimental model of septic shock caused by a MDR clinical isolate of Escherichia coli were studied. The ability of s-thanatin to bind or neutralise LPS from E. coli O111:B4 was determined using a quantitative assay kit. Male ICR mice were given an intraperitoneal (i.p.) administration of 2x10(10) colony-forming units of E. coli E79466. Following bacterial challenge, all animals were randomised to receive i.p. administration of saline, 40mg/kg ceftazidime (CAZ), or 40mg/kg CAZ+s-thanatin (10, 20 or 40mg/kg). The results showed that s-thanatin not only completely bound to the LPS (median effective concentration of 17.5microg/mL) but also improved the survival and reduced the number of inoculated bacteria in a mouse model of septic shock. s-thanatin may be an attractive candidate to develop as an anti-MDR bacterial agent.