Basic studies of the cross-linking reaction of collagen with oxazolidine and plant polyphenols

To elucidate the reaction between collagen, condensed tannins and oxazolidine, two prodeiphinidin tannins have been studied: extracts from pecan nutshell pith and myrica esculenta bark. The tannin chemistry study showed pecan tannins are copolymers of catechin and gallocatechin with a ratio 1:6 and myrica tannins are prodeiphinidins with 40% of the structural units gallated. The average molecular weight was measured by GPC and the number average molecular weight of tannins was found to be 25 00-3000. The molecular structure of oxazolidine, 1 -aza-5-ethyl- 1,3-dioxacyc1o[3 .3.0] octane, was confirmed by IR, GC-MS and ‘H-NMR spectra. From NMR, the ratio between cis and trans conformations is nearly 1:1. Complete hydrolysis of oxazolidine is slow under weakly acidic or neutral conditions. The reaction of polyphenols with oxazolidine was modelled using simple phenols phioroglucinol, pyrogallol and resorcinol; the process was followed by chromatographic and NMR methods. The results show the reaction is second order: phloroglucinol has the highest reactivity. By using catechin and gallocatechin, it was shown that the cross-linking reaction of gallocatechins can happen between an A- ring and a B-ring, while for catechin, the reaction is only at the A-ring. Polyphenol polymer studies also support this result; more oxazolidine can be reacted with prodeiphinidins. The reaction of collagen with oxazolidine was studied; the reaction sites were determined using modified collagen. Few cross-linking reactions were found, which is supported by hydrothermal isometric tension (HIT) results. The reaction between polyphenol, collagen and oxazolidine was studied by HIT and it was found that the cross-linking is between collagen and tannins, which is responsible for the high stability of treated collagen. Based on this, organic combination tanning can be explained as an effect of covalent bonding, hydrogen bonding and hydrophobic bonding together, but the high shrinkage temperature is mainly due to the covalent bonding forming a complex matrix structure around the collagen fibre

Inhibition effects of paeonol on mice bearing EMT6 breast cancer through inducing rumor cell apoptosis

Paeonol, a phenolic component from the root bark of Paeonia moutan, has been identified to possess antitumor effects on mice bearing EMT6 breast cancer in our previous studies. However, the underlying mechanisms remain unknown. In the present study the molecular mechanisms of paeonol were further investigated in EMT6 mice model. The results showed that treatment of mice with 175 and 350 mg/kg/day of paeonol significantly inhibited the growth of the EMT6 tumor in mice, and induced tumor cell apoptosis which were demonstrated by light microscopy after hematoxylin and eosin staining and apoptosis analysis by flow cytometry. In addition, compared with the control group, paeonol increased the number of tumor cells in G0/G1 phase but decreased the number of cells in S and G2/M phase. Paeonol treatment (350 mg/kg body weight) also resulted in a decrease of Bcl-2 and an increase in Bax and caspase-3 expressions, which were demonstrated by immunohistochemical and western blot analysis. These results indicate that the antitumor effects of paeonol might be associated with arresting tumor cells in the G0/G1 phase, inducing cell apoptosis and regulation of the expression of Bcl-2, Bax and activation of caspase-3

Antitumor effect of salidroside on mice bearing HepA hepatocellular carcinoma

Salidroside, a phenylpropanoid glycoside extracted from Rhodiola rosea L., has antiproliferative effects on tumour cells in mice. However it’s antitumor mechanism remains largely unknown. In this study, 4 groups of mice bearing hepatocarcinoma cells were given treatment with vehicle alone, cyclophosphamide (25 mg/kg, i.p.) and salidroside, either 100 or 200 mg/kg (p.o.) for 14 days. The morphology of tumour specimens was analysed by transmission electron microscopy. Apoptotic cells in sections of mouse tumour tissue were analysed using an in situ apoptosis kit. The expression of Bcl-2, Bax and caspase 3 mRNA were examined with RT-PCR. The results showed that the tumour weights in groups 100 or 200 mg/kg/day of salidroside were reduced significantly (45.34 and 52.48% respectively), compared to vehicle groups. Salidroside increased apoptotic cells index, e.g. in 200 mg/kg group, it was four times higher compared to the control group. Even more, treatment with salidroside decreased Bcl-2 mRNA expression and increased Bax and caspase 3 mRNA expressions. These indicated that the antitumor mechanism of salidroside may induce tumour cell apoptosis in mice by triggering the mitochondrial-dependent pathway and activation of caspase 3

The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism

Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152

Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8. Incorporation experiments using isotopically-labelled precursors confirmed the chemical structures of SCB1-3 and established those of SCB4-8

Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery

In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-L-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis, as well as inhibition of the cancer-associated metabolic enzyme Nicotinamide N-methyltransferase (NNMT)

Second-generation probes for biosynthetic intermediate capture : towards a comprehensive profiling of polyketide assembly

Malonyl carba(dethia) N-decanoyl cysteamine methyl esters and novel acetoxymethyl esters were utilised as second-generation probes for polyketide intermediate capture. The use of these tools in vivo led to the characterisation of an almost complete set of biosynthetic intermediates from a modular assembly line, providing a first kinetic overview of intermediate processing leading to complex natural product formation

Role of GntR family regulatory gene SCO1678 in gluconate metabolism in streptomyces coelicolor M145

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed

Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS — increased sensitivity resulting from reduced non-specific binding

Malaria is one of the most important parasitic diseases of man. The development of drug resistance in malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has received renewed interest as a partner drug in artemisinin-based combination therapy. To study its pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a robust, highly sensitive and accurate bioanalytical method for drug measurements in biological samples. We have developed a sensitive quantification method that covers a wide range of clinically relevant concentrations (1.5 ng/mL to 882 ng/mL) using a relatively low volume sample of 100 μL of whole blood. Total run time is 5 min and precision is within ±15% at all concentration levels. Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a HALO RP amide fused-core column using a gradient mobile phase of acetonitrile–ammonium formate and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine accurately for up to 42 days after a single oral dose in a drug-drug interaction study in healthy volunteers