Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree)

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.     

APOC1 T45S polymorphism is associated with reduced obesity indices and lower plasma concentrations of leptin and apolipoprotein C-I in aboriginal Canadians

Apolipoprotein (apo) C-I is a constituent of chylomicrons, very low density lipoprotein, and high density lipoprotein. The role of apo C-I in human metabolism is incompletely defined. We took advantage of a naturally occurring amino acid polymorphism that is present in aboriginal North Americans, namely apo C-I T45S. We assessed the hypothesis that metabolic traits, including obesity-related and lipoprotein-related traits, would differ between carriers and noncarriers of apo C-I T45S. A genotyping assay was developed for APOC1 T45S and genotypes were determined in a sample of 410 Canadian Oji-Cree subjects. The allele frequency of the apo C-I S45 allele was ∼8% in this sample. We observed the apo C-I S45 allele was significantly associated with 1) lower percent body fat (P < 0.05), 2) lower waist circumference (P = 0.058), 3) lower serum leptin levels (P < 0.05), and 4) lower plasma apo C-I levels (P < 0.0001), using a newly developed ELISA-based method. Taken together, these results suggest that at the whole human phenotype level, apo C-I is associated with the complex metabolic trait of obesity as well as with serum leptin levels.     

Secondary Hyperparathyroidism in Patients with Endemic Skeletal Fluorosis

Investigation of 20 patients with skeletal fluorosis showed that five had clear evidence of secondary hyperparathyroidism. The hyperactivity of the parathyroid glands in skeletal fluorosis in the presence of decreased solubility of the bone mineral (fluoroapatite) strongly suggests that it is a compensatory attempt to maintain a normal extracellular ionized calcium equilibrium. Further study of the parathyroid glands and of bone lesions in skeletal fluorosis is in progress.     

Application of a Newly Identified and Characterized 18-O-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins

Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS₃ from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS₃ open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (k_cat of 28 ± 3 min⁻¹ and K_m of 88 ± 16 μM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS₃ was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.Attachment of either short (C₂ to C₆) or medium (C₈ to C₁₂) acyl chains to both amine and alcohol moieties on polyketide and polypeptide natural products can represent a key step in generating the final biologically active molecule. This step is often, but not always, one of the later biosynthetic steps and is catalyzed by an acyltransferase. The corresponding gene is typically associated with the polyketide or polypeptide biosynthetic gene cluster. Despite the importance of this step, a relatively small number of these acyltransferases from actinomycetes have been identified, and very few have been fully characterized (2, 10, 15, 18).Studies of biosynthetic processes where there is an acylation of a polyketide chain, have indicated that the enzymes have various degrees of promiscuity for the carboxylic acid substrates. For instance, genetic evidence has shown that mdmB and acyA encode 3-O-acyltransferases which transfer either acetyl or propionyl groups to position 3 in 16-membered macrolides such as midecamycin, spiramycin, carbomycin, and tylosin (3, 10). The asm19 gene product has been identified as the 3-O-acyltransferase which catalyzes the attachment of the biologically essential acyl group in the macrocyclic ansamitocins (18). The asm19 mutant accumulates an N-demethyl-4,5-desepoxymytansinol, indicating that acylation of the macrocycle precedes N methylation and epoxidation. Escherichia coli cell extracts containing a recombinant Asm19 protein have been shown to catalyze acylation of this mytansinol intermediate using a range of short straight- and branched-chain acyl coenzyme A (CoA) thioesters (C₂ to C₅). Finally, LovD, which catalyzes the acylation of the C-8 hydroxyl group of monacolin J to yield the natural product lovastatin, a pharmaceutically important fungal polyketide product produced by Aspergillus nidulans, has been characterized (30). This enzyme is able to utilize a wide range of different acyl donors activated as CoA, N-acetylcysteamine (NAC), or methyl thioglycolate esters and thus offers an economically attractive route for generating novel lovasotatin analogs for treatment of hypercholesterolemia.One or multiple O-acyltransferases have been implied to be involved in the post-polyketide synthase tailoring steps leading to a series of natural products known as the phoslactomycins (PLMs) (Fig. ​(Fig.1)1) (6). These compounds (also known as leustroducsins [LSNs], phospholines, and phosphazomycins) have been isolated from various actinomycetes, and their structures are all identical with the exception of the acyl substituent at C-18 (4, 5, 12, 13, 20, 27, 28). Streptomyces sp. strain HK 803 produces at least five such acylated analogs (PLM A and PLMs C to F) (Fig. ​(Fig.1)1) as well as PLM B, in which the C-18 hydroxyl substituent is absent (4, 5, 27).Open in a separate windowFIG. 1.Proposed biosynthetic relationship between PLM products made by Streptomyces sp. strain HK 803. A cytochrome P450 monooxygenase (PlmS₂) catalyzes C-18 hydroxylation of PLM B to generate PLM G, which is subsequently 18-O acylated by PlmS₃.These natural products have been isolated based on their potent activity (as low as 0.008 μg/ml) against some phytopathogenic fungi (27, 28). The compounds also have relatively weak antitumor activity (50% inhibitory concentration of 2 to 3 μg/ml against L1210, P38,8, and El-4 cell lines) (19) which may arise from their activity as selective inhibitors of protein phosphatase 2A. (26). These natural products also show induction of a colony-stimulating factor (12) via NF-κB activation and thrombopoiesis (14). This array of promising biological activities has stimulated research into the field of PLMs for treatments of various diseases. Low yields and the presence of multiple acylated products from fermentations have provided a barrier to this work, and circuitous routes to obtaining individual compounds have been described. For instance, there have been no reports of any actinomycetes which produce PLM G (LSN H) (Fig. ​(Fig.1),1), in which the C-18 hydroxyl residue is present but not acylated, and this compound has been reported to be obtained only by cleavage of the acyl groups of a mixture of other PLMs and LSNs using porcine liver esterase (24). A multistep synthetic route to selectively acylate PLM G with 6-methyloctanoic acid (producing LSN B) has also been described (17).Recently the entire 75-kbp Plm biosynthetic gene cluster has been cloned, sequenced, and analyzed (21) and has provided an opportunity to study the enzymatic hydroxylation and acylation processes which give rise to the range of PLM products. Deletion of the plmS₂ open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases, has resulted in an NP1 mutant producing only PLM B (Fig. ​(Fig.1)1) (21). The plmS₂ ORF has been expressed as an N-terminally polyhistidine-tagged protein in Streptomyces coelicolor, and the purified protein has been shown to catalyze conversion of PLM B to PLM G (7). This work in conjunction with other studies (1, 23) has led to a proposal that the final two biosynthetic steps involve hydroxylation of PLM B (to give PLM G) and subsequent acylation with a broad range of acyl-CoA substrates (to give PLM A and PLMs C to F). The acylation step is required for potent antifungal activity of the PLMs. Initial analysis of the PLM biosynthetic gene cluster (21) did not reveal a candidate gene or genes whose products might be responsible for this acylation.Here we identify the plmS₃ gene product as the singular acyltransferase in Streptomyces sp. strain HK 803 responsible for C-18 acylation of PLM G. Generation of a plmS₃ deletion mutant results in selective production of PLM G, supporting the proposed role of this gene product and providing the first direct fermentation method to access this intermediate. The 921-bp plmS₃ ORF was cloned and expressed as an N-terminally polyhistidine-tagged protein in E. coli, and the recombinant purified protein was shown to catalyze acylation of PLM G with isobutyryl-CoA, 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA to give PLM A, PLM C, and PLM E, respectively. This efficient one-step enzymatic process offers an attractive alternative to the multistep synthetic process for selective acylation of PLM G. PlmS₃ was also shown to catalyze esterification of PLM G with CoA and NAC thioesters with a remarkably wide range of various saturated, unsaturated, and aromatic carboxylic acids and thus provides an efficient chemoenzymatic route to new PLM analogs.     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.     

Genetic manipulation of the biosynthetic process leading to phoslactomycins, potent protein phosphatase 2A inhibitors

Phoslactomycins (PLMs) represent an unusual structural class of natural products secreted by various streptomycetes, containing an α,β-unsaturated δ-lactone, an amino group, phosphate ester, conjugated diene and a cyclohexane ring. Phosphazomycins, phospholines and leustroducsins contain the same structural moieties, varying only in the acyl substituent at the C-18 hydroxyl position. These compounds possess either antifungal or antitumor activities or both. The antitumor activity of the PLM class of compounds has been attributed to a potent and selective inhibition of protein phosphatase 2A (PP2A). The cysteine-269 residue of PP2Ac-subunit has been shown to be the site of covalent modification by PLMs. In this article, we review previous work on the isolation, structure elucidation and biological activities of PLMs and related compounds and current status of our work on both PLM stability and genetic manipulation of the biosynthetic process. Our work has shown that PLM B is surprisingly stable in solution, with a pH optimum of 6. Preliminary biosynthetic studies utilizing isotopically labeled shikimic acid and cyclohexanecarboxylic acid (CHC) suggested PLM B to be a polyketide-type antibiotic synthesized using CHC as a starter unit. Using a gene (chcA) from a set of CHC-CoA biosynthesis genes from Streptomyces collinus as a probe, a 75 kb region of 29 ORFs encoding PLM biosynthesis was located in the genome of Streptomyces sp. strain HK803. Analysis and subsequent manipulation of plmS ₂ and plmR ₂ in the gene cluster has allowed for rational engineering of a strain that produces only one PLM analog, PLM B, at ninefold higher titers than the wild type strain. A strain producing PLM G (the penultimate intermediate in PLMs biosynthesis) has also been generated. Current work is aimed at selective in vitro acylation of PLM G with various carboxylic acids and a precursor-directed biosynthesis in a chcA deletion mutant with the aim of generating novel PLM analogs.     

Evaluation of anti-quorum sensing activity of silver nanowires

A menace of antimicrobial resistance with growing difficulties in eradicating clinical pathogens owing to the biofilm has prompted us to take up a facile aqueous-phase approach for the synthesis of silver nanowires (SNWs) by using ethylene glycol as a reducing agent and polyvinylpyrrolidone (PVP) as a capping agent. This synthesis is a reflux reaction seedless process. The obtained SNWs were about 200–250 nm in diameter and up to 3–4 μm in length. The SNWs were characterized by field emission scanning electron microscopy, transmission electron microscopy, UV–Vis spectroscopy, and X-Ray powder diffraction, and the chemical composition of the sample was examined by energy dispersive X-ray spectrum. The SNWs did not show an antibacterial activity against test organisms, Bacillus subtilis NCIM 2063 and Escherichia coli NCIM 2931; however, it showed a promising property of a quorum sensing-mediated inhibition of biofilm in Pseudomonas aeruginosa NCIM 2948 and violacein synthesis in Chromobacterium violaceum ATCC 12472, which is hitherto unattempted, by polyol approach.