Infiltration of Natural Caries Lesions in Relation to Their Activity Status and Acid Pretreatment in vitro

This study aimed at testing how active and inactive enamel caries lesions differ by their degree of resin infiltration, and whether the choice of acid pretreatment plays a crucial role. Four examiners assessed 104 human molars and premolars with noncavitated enamel lesions and classified them as 'active' or 'inactive' using the Nyvad criteria. Forty-five teeth were included in this study after independent unanimous lesion activity assessment. Lesions were cut perpendicularly into 2 halves. Each half lesion was pretreated with either 15% hydrochloric acid or 35% phosphoric acid. The lesions were infiltrated after staining with rhodamine isothiocyanate. Thin sections of 100 µm were prepared and the specimens were bleached with 30% hydrogen peroxide. The specimens were then counterstained with sodium fluorescein, subjected to confocal laser scanning microscopy and analyzed quantitatively. Outcome parameters were maximum and average infiltration depths as well as relative penetration depths and areas. In active lesions no significant difference of percentage maximum penetration depth and percentage average penetration depth between lesions pretreated with hydrochloric or phosphoric acid could be observed. In inactive lesions, however, phosphoric acid pretreatment resulted in significantly lower penetration compared to hydrochloric acid pretreatment. Surface conditioning with hydrochloric acid led to similar infiltration results in active and inactive lesions. Moreover, inactive lesions showed greater variability in all assessed infiltration parameters than did active lesions. In conclusion, caries lesion activity and acid pretreatment both influenced the infiltration. The use of phosphoric acid to increase permeability of the surface layer of active lesions should be further explored.

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Pancreatic stone protein is highly increased during posttraumatic sepsis and activates neutrophil granulocytes

OBJECTIVES: The level of pancreatic stone protein/regenerating protein (PSP/reg), a secretory protein produced in the pancreas, increases dramatically during pancreatic disease. However, after stress (e.g., anesthesia), PSP/reg levels are increased transiently in animals without pancreatic injury. Therefore, we aimed to determine whether PSP/reg is an acute-phase protein after nonpancreatic trauma. PATIENTS: Eighty-three polytraumatic patients without pancreatic damage. MEASUREMENTS AND MAIN RESULTS: We compared serum PSP/reg levels from polytraumatic patients without pancreatic damage with those in healthy controls (n = 38). C-reactive protein, interleukin-6, procalcitonin, and leukocyte numbers were also compared. The expression of CD62L and CD11b on neutrophils after exposure to PSP/reg was analyzed by flow cytometry. Thirty-three patients (39%) developed sepsis, 32 (38%) had local infections, and 18 (21%) had no infections. At admission, PSP/reg serum levels (10.2 [6.2-14.5] ng/mL; median [interquartile range]) were comparable with those in healthy controls (10.4 [7.5-12.3] ng/mL). During hospital stay, PSP/reg levels were elevated significantly in patients with sepsis (146.4 ng/mL) and in patients with infections (111.4 ng/mL) compared with patients without infections (22.8 ng/mL). Furthermore, binding of fluorescein isothiocyanate-labeled recombinant PSP/reg to human neutrophils was demonstrated. Recombinant PSP/reg elicited a dose-dependent shedding of L-selectin (CD62L) and upregulation of beta2-integrin (CD11b) in neutrophils, which indicates that PSP/reg activates neutrophils. CONCLUSIONS: We conclude that PSP/reg is up-regulated in blood after trauma, and the PSP/reg level is related to the severity of inflammation. Furthermore, PSP/reg binds to and activates neutrophils. Therefore, PSP/reg might be an acute-phase protein that could serve as a marker for posttraumatic complications.

Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABA receptors

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of alpha(1)S205C and alpha(1)T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (-NCS). We also provide new data that identify alpha(1)H101C and alpha(1)N102C as exclusive sites of the reaction of a diazepam derivative where the -Cl atom is replaced by a -NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of alpha(1)beta(2)gamma(2) receptors.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-benzylated L-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

Liquid-Crystalline Dendrimers Designed by Click Chemistry

Liquid-crystalline dendrimers have been prepared from second-generation Percec-type poly(benzyl ether) dendrons or second-generation poly(aryl ester) dendrons carrying cyanobiphenyl mesogens. The Janus dendrimer, which combines the two types of dendromesogens, has also been synthesized. Those compounds have been prepared under copper-catalyzed azide–alkyne cycloaddition conditions. The mesomorphic properties have been studied by thermal analysis (POM, DSC) and small-angle X-ray scattering. Smectic A, nematic, and columnar phases have been observed depending on the dendritic building blocks. The click reaction has proven to be a powerful and elegant synthetic tool for the design of complex dendritic liquid-crystalline architectures.