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PURPOSE: Red grape pomace extract (oenocyanin) is a cheap and rich source of anthocyanins, the agents suggested to possess cancer chemopreventive properties. Here the hypothesis was tested that oenocyanin added to the diet can interfere with intestinal adenoma development in the Apc(Min) mouse, a model of intestinal carcinogenesis linked to an Apc mutation. METHODS: Mice received oenocyanin (0.3%) in their diet until week 16, when adenoma number and burden were recorded. Expression of Akt and ERK proteins was studied by Western blot in adenomas to discover effects of anthocyanins on cellular signalling via the PI3 and MAP kinase pathways. Levels of anthocyanins were measured by HPLC with visible spectroscopic or mass spectrometric detection. RESULTS: In mice which had consumed oenocyanin, overall adenoma burden was halved and adenoma number was marginally reduced when compared with mice on control diet. The proliferation index in colonic adenomatous crypts, as reflected by Ki-67 staining, was significantly decreased from 88.14% in control mice to 75.6+/-4% in mice on oenocyanin (P=0.014). Expression of Akt in small intestinal adenomas from Apc(Min) mice on oenocyanin was reduced by 54% (P=0.003), when compared to controls. Oenocyanin anthocyanins and glucuronide metabolites were found in the urine and intestine but not in plasma. CONCLUSIONS: The results suggest that oenocyanin may be a viable and economical alternative to anthocyanin-rich berry extracts for chemopreventive intervention. Akt and pErk might be suitable biomarkers of anthocyanin target organ efficacy. [ABSTRACT FROM AUTHOR]

There has been growing attention on the effect of COVID-19 on white-matter microstructure, especially among those that self-isolated after being infected. There is also immense scientific interest and potential clinical utility to evaluate the sensitivity of single-shell diffusion magnetic resonance imaging (MRI) methods for detecting such effects. In this work, the performances of three single-shell-compatible diffusion MRI modeling methods are compared for detecting the effect of COVID-19, including diffusion-tensor imaging, diffusion-tensor decomposition of orthogonal moments and correlated diffusion imaging. Imaging was performed on self-isolated patients at the study initiation and 3-month follow-up, along with age- and sex-matched controls. We demonstrate through simulations and experimental data that correlated diffusion imaging is associated with far greater sensitivity, being the only one of the three single-shell methods to demonstrate COVID-19-related brain effects. Results suggest less restricted diffusion in the frontal lobe in COVID-19 patients, but also more restricted diffusion in the cerebellar white matter, in agreement with several existing studies highlighting the vulnerability of the cerebellum to COVID-19 infection. These results, taken together with the simulation results, suggest that a significant proportion of COVID-19 related white-matter microstructural pathology manifests as a change in tissue diffusivity. Interestingly, different b-values also confer different sensitivities to the effects. No significant difference was observed in patients at the 3-month follow-up, likely due to the limited size of the follow-up cohort. To summarize, correlated diffusion imaging is shown to be a viable single-shell diffusion analysis approach that allows us to uncover opposing patterns of diffusion changes in the frontal and cerebellar regions of COVID-19 patients, suggesting the two regions react differently to viral infection., (© 2023 The Authors. Human Brain Mapping published by Wiley Periodicals LLC.)

Management of mangrove ecosystems is complex, given that mangroves are both terrestrial and marine, often cross regional or national boundaries, and are valued by local stakeholders in different ways than they are valued on national and international scales. Thus, mangrove governance has had varying levels of success, analyzed through concepts such as principles of good governance and procedural justice in decision-making. Although there is substantial research on case studies of mangrove management, global comparisons of mangrove governance are lacking. This research aims to fill this gap by comparing relationships among qualities of governance across mangrove social-ecological systems worldwide. Through a systematic literature search and screening process, we identified 65 articles that discussed mangrove governance and conservation. Case studies in these articles, drawn from 39 countries, were categorized as top-down, bottom-up, or comanaged and thematically coded to assess the influence of eight principles of good governance in mangrove conservation success. Across all three governance systems, the principles of legitimacy, fairness, and integration were most important in determining conservation success or failure. These principles are closely related to the concept of procedural justice, highlighting the importance of stakeholder inclusion throughout all stages of mangrove management. Thus, we recommend clearly defined roles for all governance actors, transparent communication of policy development to stakeholders, fairness in both process and outcome, and careful consideration of sustainable access to conservation resources., (© 2021 Society for Conservation Biology.)

Inducible systems for gene expression emerge as a new class of artificial vectors offering temporal and spatial exogenous control of gene expression. However, most inducible systems are less efficient in vivo and lack the target-organ specificity. In the present study, we have developed and optimized an oligonucleotide-based inducible system for the in vivo control of transgenes in the liver. We generated a set of simple, inducible plasmid-vectors based on the addition of four units of liver-specific miR-122 target sites to the 3'untranslated region of the gene of interest. Once the vector was delivered into hepatocytes this modification induced a dramatic reduction of gene expression that could be restored by the infusion of an antagomir for miR-122. The efficiency of the system was tested in vivo, and displayed low background and strong increase in gene expression upon induction. Moreover, gene expression was repeatedly induced even several months after the first induction showing no toxic effect in vivo. By combining tissue-specific control elements with antagomir treatment we generated, optimized and validated a robust inducible system that could be used successfully for in vivo experimental models requiring tight and cyclic control of gene expression.

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

In cell culture were compared the different release rates of anthocyanins from a bilberry pomace extract encapsulated either in food grade whey protein-based matrix capsules (WPC) or in pectin amid-based hollow spherical capsules (PHS). The impact of the formulations on typical anthocyanin-associated biological end points such as inhibition of the epidermal growth factor receptor (EGFR) and suppression of cell growth in HT29 colon carcinoma cells was assessed. The purpose was to find whether the release rates are sufficient to maintain biological activity and whether encapsulation affected EGFR inhibitory and growth suppressive properties of the extract. Even though anthocyanin release from extract-loaded capsules was proven under cell culture conditions, the inhibitory potential toward the EGFR was diminished. However, nonencapsulated extract as well as both extract-loaded encapsulation systems diminished the growth of HT29 cells to a comparable extent. The loss of EGFR inhibitory properties by encapsulation despite anthocyanin release indicates substantial contribution of other further constituents not monitored so far. Taken together, both applied encapsulation strategies allowed anthocyanin release and maintained biological activity with respect to growth inhibitory properties. However, the loss of EGFR inhibitory effects emphasizes the need for biological profiling to estimate process-induced changes of plant constituent's beneficial potencies.

Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 μM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 ∼ 100 μg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 ∼ 10 μg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.

This study investigated Nrf2-activating properties of a coffee blend combining raw coffee bean constituents with 5-O-caffeoylquinic acid (CGA) as a lead component with typical roasting products such as N-methylpyridinium (NMP). In cell culture (HT29) the respective coffee extract (CN-CE) increased nuclear Nrf2 translocation and enhanced the transcription of ARE-dependent genes as exemplified for NAD(P)H:quinone oxidoreductase and glutathione-S-transferase (GST)A1, reflected in the protein level by an increase in GST enzyme activity. In a pilot human intervention study (29 healthy volunteers), daily consumption of 750 mL of CN-coffee for 4 weeks increased Nrf2 transcription in peripheral blood lymphocytes on average. However, the transcriptional response pattern of Nrf2/ARE-dependent genes showed substantial interindividual variations. The presence of SNPs in the Nrf2-promoter, reported recently, as well as the detection of GSTT1*0 (null) genotypes in the study collective strengthens the hypothesis that coffee acts as a modulator of Nrf2-dependent gene response in humans, but genetic polymorphisms play an important role in the individual response pattern.

Oligonucleotides (ONs) and more particularly siRNAs are promising drugs but their pharmacokinetics and biodistribution are widely unknown. Positron Emission Tomography (PET) using fluorine-18 is a suitable technique to quantify these biological processes. Click chemistry (Huisgen cycloaddition) is the current method for labeling siRNA. In order to study the influence of a linker bearing by [(18)F] labeled ONs, on the in vivo pharmacokinetic and metabolism, we have developed two modified ONs by two new linkers. Here we report the synthesis of two alkyne-bearing linkers, the incorporation onto a ONs and the conjugation by click chemistry with a [(18)F] prosthetic group., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Nrf2/ARE pathway is a major cellular defense mechanism that prevents damage by reactive oxygen species through induction of antioxidative phase II enzymes. However, the activity of the Nrf2/ARE system is not uniform with variability in response presumed to be dependent on the Nrf2 genotype. We recently completed a pilot human coffee intervention trial with healthy humans, where large interindividual differences in the antioxidative response to the study coffee were examined. Here, we address the question whether differences in the modulation of Nrf2 gene transcription, assessed as an induction of Nrf2 gene transcription by Q-PCR, might be correlated with specific Nrf2 genotypes. To date, nine single nucleotide polymorphisms (SNPs) have been identified in the Nrf2 (NFE2L2) gene. Two of these, the -617C/A and -651G/A SNPs are located within the promoter region and have previously been reported to influence the activity of the Nrf2/ARE pathway by reducing Nrf2 transcriptional activity. Sequencing of the critical Nrf2 gene promoter region not only confirmed the existence of these SNPs within the participants of the trial at the expected frequency (33% carrying the -617C/A, 17% the -651G/A and 56% the -653A/G SNP) but also indicated reduced Nrf2 gene transcription associated with a normal diet if the SNPs at position -617, -651 or -653 were present. Of note, the data also indicated the study coffee increased Nrf2 gene transcription even in SNP carriers. This further highlights the relevance of genotype-dependent induction of Nrf2 gene transcription that appears to be largely influenced by dietary factors.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recently, the coffee constituents 5-O-caffeoylquinic acid (CGA) and N-methylpyridinium (NMP) were identified as inducers of the Nrf2/antioxidant-response element (ARE) detoxifying pathway under cell-culture condition. To study the impact of CGA and NMP on the Nrf2-activating properties of a complex coffee beverage, two different model coffees were generated by variation of the roasting conditions: a low-roast coffee rich in CGA and a heavy-roast low in CGA but containing high levels of NMP. Activation of the Nrf2/antioxidant-response element pathway was monitored in vitro and in vivo., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined., Methods and Results: The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells., Conclusion: These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The alkyne-azide Cu(I)-catalyzed Huisgen cycloaddition, a click-type reaction, was used to label a double-stranded oligonucleotide (siRNA) with fluorine-18. An alkyne solid support CPG for the preparation of monostranded oligonucleotides functionalized with alkyne has been developed. Two complementary azide labeling agents (1-(azidomethyl)-4-[(18)F]fluorobenzene) and 1-azido-4-(3-[(18)F]fluoropropoxy)benzene have been produced with 41% and 35% radiochemical yields (decay-corrected), respectively. After annealing with the complementary strand, the siRNA was directly labeled by click chemistry with [(18)F]fluoroazide to produce the [(18)F]-radiolabeled siRNA with excellent radiochemical yield and purity.

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-γ-D-Glu) amidated by benzylamine on the γ-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-γ-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-γ-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the ε-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli. The peptide D-Glu-delta-m-A(2)pm-D-Ala-m-A(2)pm-D-Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd-endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed.

Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.

Delphinidin has been reported to inhibit EGFR signalling. To determine whether other receptor tyrosine kinases (RTKs) are also influenced by delphinidin, we examined its ability to inhibit the kinase activity of EGFR, ErbB2, VEGFR-2, VEGFR-3 and IGF1R in a cell-free test system. We found that delphinidin strongly inhibited the protein tyrosine kinase activity of all tested RTKs at low micromolar concentrations. In A431 and PAE cells, ligand-induced phosphorylation of the receptors was also potently suppressed, with a preference for the suppression of the activity of ErbB3 (IC(50) approximately 100 nM) and VEGFR-3 (IC(50) < 50 microM). Thus the inhibition of RTKs by delphinidin is not limited to cell-free assays but is also of relevance in the cellular context. The results indicate that delphinidin acts as a broad-spectrum inhibitor of RTKs. Given the crucial role of the receptors in tumour growth and metastasis, we conclude that delphinidin has the potential to act directly against tumour cells as well as to interfere with key tumour-host interactions, although the suitability of delphinidin as a drug in cancer management may be compromised by its limited stability. Nevertheless, delphinidin may represent a novel lead compound for the development of chemopreventative and chemotherapeutic intervention strategies.

Anthocyanins, plant pigments in fruits and berries, have been shown to delay cancer development in rodent models of carcinogenesis, especially those of the colorectal tract. Anthocyanins and anthocyanidins, their aglycons, especially cyanidin and delphinidin, have been subjected to extensive mechanistic studies. In cells in vitro, both glycosides and aglycons engage an array of anti-oncogenic mechanisms including anti-proliferation, induction of apoptosis and inhibition of activities of oncogenic transcription factors and protein tyrosine kinases. Anthocyanins and anthocyanidins exist as four isomers, interconversion between which depends on pH, temperature and access to light. Anthocyanidins are much more prone to avid chemical decomposition than the glycosides, and they only survive for minutes in the biophase. These pharmaceutical issues are very important determinants of the suitability of these flavonoids for potential development as cancer chemopreventive drugs, and they have hitherto not received adequate attention. In the light of their robust cancer chemopreventive efficacy in experimental models and their superior stability as compared to that of the aglycons, the anthocyanins seem much more suitable for further drug development than their anthocyanidin counterparts.

Two standardized anthocyanin-rich mixtures were investigated for their ability to inhibit the receptor tyrosine kinases (RTKs) EGFR, ErbB2, ErbB3, VEGFR-2, and VEGFR-3. Both mixtures reduced the kinase activity of recombinant kinase domains of each RTK at concentrations or=50 microg/mL. These results indicate that anthocyanin-rich mixtures can inhibit RTKs with low specificity. The rank order of inhibitory efficacy against the tested RTKs in intact cells was VEGFR-3 >> VEGFR-2 > ErbB3 > EGFR > ErbB2. Considering the important role of RTKs in carcinogenesis, their inhibition by anthocyanin-rich mixtures suggests that they may serve as biomarkers of the pharmacological efficacy of anthocyanins in future chemoprevention experiments and in clinical intervention studies.

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T(P1)R.

In the present study, delphinidin was found to suppress the phosphorylation of the epidermal growth factor receptor (EGFR) within human tumour cells (human colon carcinoma cell line (HT29), human vulva carcinoma cell line (A431)), albeit less effective than the flavonol quercetin. The higher potency of quercetin was also observed downstream on the level of the mitogen-activated protein kinase (MAPK) cascade. In addition, delphinidin, quercetin and (-)-epigallocatechin-3-gallate (EGCG) were found to suppress the phosphorylation of the ErbB2 receptor, with delphinidin exhibiting the strongest inhibitory properties. Their potency to suppress the ErbB2 receptor phosphorylation can be summarised as delphinidin > EGCG > quercetin. The effectiveness of delphinidin against the EGFR and the ErbB2 receptor was comparable, indicating a broader spectrum of activity against receptor tyrosine kinases. At low micromolar concentrations delphinidin showed some preference towards the ErbB2 receptor. In summary, quercetin and delphinidin appear to differ in their activity profile towards the ErbB receptor family members. Whereas quercetin was most effective against the EGFR, delphinidin exhibited some preference towards the ErbB2 receptor.

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of beta-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of beta-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of beta-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.