Novel benzyl-substituted N-heterocyclic carbene-silver acetate complexes: synthesis, cytotoxicity and antibacterial studies

From the reaction of 1-methylimidazole (1a), 4,5-dichloro-1H-imidazole (1b(I)) and 1-methylbenzimidazole (1c) with p-cyanobenzyl bromide (2a), non-symmetrically substituted N-heterocyclic carbene (NHC) [(3a-c)] precursors, 5,6-dimethyl-1H-benzimidazole (1d) and 4,5-diphenyl-1H-imidazole (1e) with p-cyanobenzyl bromide (2a) and benzyl bromide (2b), symmetrically substituted N-heterocyclic carbene (NHC) [(3d-f)] precursors were synthesised. These NHC-precursors were then reacted with silver(i) acetate to yield the NHC-silver complexes (1-methyl-3-(4-cyanobenzyl)imidazole-2-ylidene)silver(i)acetate (4a), (4,5-dichloro-1-(4-cyanobenzyl)-3-methyl)imidazole-2-ylidene)silver(i)acetate (4b), (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene)silver(i)acetate (4c), (1,3-bis(4-cyanobenzyl)5,6-dimethylbenzimidazole-2-ylidene) silver(i) acetate (4d), (1,3-dibenzyl-5,6-dimethylbenzimidazole-2-ylidene) silver(i) acetate (4e) and (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(i) acetate (4f) respectively. Three NHC-precursors 3c-e and four NHC-silver complexes 4b and 4d-f were characterised by single crystal X-ray diffraction. Preliminary in vitro antibacterial activity of the NHC-precursors and NHC-silver complexes was investigated against Gram-positive bacteria Staphylococcus aureus, and Gram-negative bacteria Escherichia coli using the qualitative Kirby-Bauer disk-diffusion method. NHC-silver complexes have shown very high antibacterial activity compared to the NHC-precursors. All six NHC-silver complexes were tested for their cytotoxicity through MTT based in vitro tests on the human renal-cancer cell line Caki-1 in order to determine their IC?? values. NHC-silver complexes 4a-f were found to have IC?? values of 6.2 (±1.0), 7.7 (±1.6), 1.2 (±0.6), 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) μM, respectively. These values represent improved cytotoxicity against Caki-1, most notably for 4c, which is a three times more cytotoxic than cisplatin (IC?? value = 3.3 μM) itself.     

TGF-beta1 gene transfer to the mouse colon leads to intestinal fibrosis

Crohn's disease (CD) is a chronic, relapsing inflammatory bowel disease, characterized by transmural inflammation. In CD, the recurrent inflammatory injury and tissue repair that occurs in the intestine can progress uncontrollably, leading to the proliferation of mesenchymal cells as well as fibrosis, characterized by excessive extracellular matrix deposition. These processes thicken the bowel wall, reducing flexibility, and often culminate in obstructive strictures. Because no effective measures are currently available to specifically treat or prevent intestinal stricturing, we sought to gain a better understanding of its pathogenesis by developing a mouse model of intestinal fibrosis. Because transforming growth factor (TGF)-beta1 can mediate both fibrosis and mesenchymal cell proliferation; we studied the effects of delivering adenoviral vectors encoding spontaneously active TGF-beta1 into the colons of mice. We first demonstrated that enema delivery of marker adenoviral vectors led to the transfection of the colonic epithelium and transient transgene expression. Histologically, control vectors caused an acute inflammatory response, involving the recruitment of neutrophils and mononuclear cells into the colonic lamina propria; however, infection caused little if any fibrosis. In contrast, the TGF-beta1 vector caused a more severe and prolonged inflammatory response as well as localized collagen deposition, leading to severe and progressive fibrosis. This was accompanied by the emergence of cells with a myofibroblast phenotype. Ultimately the fibrosis resulted in many of the TGF-beta1-transfected mice developing profound colonic obstruction. Through adenoviral gene transfer technology, we describe a novel mouse model of colitis and implicate TGF-beta1 in the pathogenesis of obstructive intestinal fibrosis.     

A novel transposon for functional expression of DNA libraries

Environmental DNA libraries are important sources for novel biocatalyst genes but activity screening for relevant enzymes is often inefficient. Therefore, we have constructed the transposon MuExpress which randomly integrates in vitro into existing bacterial artificial chromosome (BAC) or cosmid libraries and permits the inducible expression of its flanking regions in both directions. Furthermore, this transposon allows the bidirectional sequencing of the respective clones starting from unique primer binding sites.     

Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.     

Interactions among flavonoids of propolis affect antibacterial activity against the honeybee pathogen Paenibacillus larvae

Propolis is derived from plant resins, collected by honeybees (Apis mellifera) and renown for its antibacterial properties. Here we test the antibacterial effects of ethanolic extracts of propolis from different origins on Paenibacillus larvae, the bacterial pathogen that causes American Foulbrood, a larval disease that can kill the honeybee colony. All tested propolis samples inhibited significantly the growth of P. larvae tested in vitro. The extracts showed major differences in the content of total flavonoids (ranging from 2.4% to 16.4%) and the total polyphenols (ranging between 23.3% and 63.2%). We found that it is not only the content of compounds in propolis, which influences the strength of antimicrobial effects but there is also a significant interaction effect among flavonoids of the propolis extracts. We propose that interaction effects among the various chemical compounds in propolis should be taken into account when considering the antibacterial effects against honeybee pathogens.     

Endurance Training Per Se Increases Metabolic Health in Young,Moderately Overweight Men

Health benefits of physical activity may depend on a concomitant weight loss. In a randomized, controlled trial, we compared the effects of endurance training with or without weight loss to the effect of weight loss induced by an energy‐reduced diet in 48 sedentary, moderately overweight men who completed a 12‐week intervention program of training (T), energy‐reduced diet (D), training and increased diet (T‐iD), or control (C). An energy deficit of 600 kcal/day was induced by endurance training or diet in T and D and a similar training regimen plus an increased dietary intake of 600 kcal/day defined the T‐iD group. Primary end point was insulin sensitivity as evaluated by HOMA‐IR (mainly reflecting hepatic insulin sensitivity) and hyperinsulinemic, isoglycemic clamps (primarily reflecting peripheral insulin sensitivity). Body mass decreased in T and D by 5.9 ± 0.7 and 5.3 ± 0.7 kg, respectively, whereas T‐iD and C remained weight stable. Total and abdominal fat mass were reduced in an additive manner in the T‐iD, D, and T groups by 1.9 ± 0.3/0.2 ± 0.1, 4.4 ± 0.7/0.5 ± 0.1, and 7.7 ± 0.8/0.9 ± 0.1 kg, respectively. HOMA‐IR was improved in T, D, and T‐iD, whereas insulin‐stimulated glucose clearance and suppression of plasma nonesterified fatty acids (NEFAs) were increased only in the two training groups. Thus, loss of fat mass (diet or training induced) improves hepatic insulin sensitivity, whereas peripheral insulin sensitivity in skeletal muscle and adipose tissue is increased by endurance training only. This demonstrates that endurance training per se increases various metabolic health parameters and that endurance training should preferably always be included in any intervention regimen for improving metabolic health in moderately overweight men.