Synthesis of peptide-based polymers by microwave-assisted cycloaddition backbone polymerization

The optimized reaction conditions for the Cu(I)-catalyzed N-->C polymerization of azido-phenylalanyl-alanyl-propargyl amide to yield either high molecular weight linear polymers or medium-sized cyclic polymers is described. These reaction conditions will be applied to tailor the synthesis, properties, and structure of biologically relevant peptide-based biopolymers.     

Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E(-/-) mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E(-/-) mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.     

Visualizing single DNA-bound proteins using DNA as a scanning probe

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.     

Microscopic diagnosis of vulvovaginal candidiasis in stained vaginal smears by Dutch general practitioners

OBJECTIVE: To determine the accuracy of the microscopic diagnosis of vulvovaginal candidiasis (presence of [pseudo] hyphae and blastospores) in stained vaginal smears in clinical practice. STUDY DESIGN: General practitioners trained in diagnosing vulvovaginal candidiasis performed microscopy of 324 stained vaginal smears. These smears were sent to the pathologist for confirmation of the microscopic diagnosis of the clinician; cytologic diagnosis by the pathologist was considered the gold standard. RESULTS: In 104 of the 342 cases Candida was established by the pathologist. The clinicians made 24 false positive and 50 false negative diagnoses of Candida. Sensitivity and specificity of the microscopic diagnoses of the clinicians were 52% and 89%, respectively. The most frequent reason for a false positive diagnosis was presence of hairs, whereas the most frequent reason for a false negative diagnosis was understaining of the smear. CONCLUSION: This study shows that even in stained smears it is difficult for clinicians to recognize blastospores and (pseudo)hyphae. Efforts are clearly needed to improve the quality of the clinical diagnosis of vulvovaginal candidiasis.     

Volatile science? Metabolic engineering of terpenoids in plants

Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites.     

Stimulation of skin's energy metabolism provides multiple benefits for mature human skin

As an organism ages, there is a decline in mitochondrial function and cellular energy balance. This decline is both accelerated by and can cause the formation of reactive oxygen species (ROS) that damage nuclear and mitochondrial DNA, lipid membranes as well as structural and catalytic proteins, especially those involved in energetic pathways of cells. Further, ROS have also been linked to some of the detrimental skin changes that occur as a result of photoaging. We have previously shown that levels of Coenzyme Q10 (CoQ10), a component of the respiratory chain in mitochondria, are reduced in skin cells from aging donors, and that topical supplementation can ameliorate processes involved in skin aging. Creatine is another important component of the cellular energy system and phosphocreatine, its phosphorylated form, functions as a reservoir for high energy phosphates. Unfortunately the creatine system and thus the energy storage mechanism in skin are negatively affected by aging and conditions of oxidative stress. This article reviews some of our in vivo data about the synergistic effects of combining a stabilized form of Creatine with CoQ10 and clearly depicts their beneficial effects as active ingredients in topical formulations.     

Activation of CFTR by ASBT-mediated bile salt absorption

In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state.     

Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat

Two genes encoding family 11 endo-beta-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli. The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying different expression parameters, including host strain and temperature during induction. Both recombinant endoxylanases showed a temperature optimum around 35 degrees C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively). For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP. Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I. The insensitivity of both F. graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi. As F. graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence.