Novel approach for genotyping varicella-zoster virus strains from Germany

In this study, we present a novel genotyping scheme to classify German wild-type varicella-zoster virus (VZV) strains and to differentiate them from the Oka vaccine strain (genotype B). This approach is based on analysis of four loci in open reading frames (ORFs) 51 to 58, encompassing a total length of 1,990 bp. The new genotyping scheme produced identical clusters in phylogenetic analyses compared to full-genome sequences from well-characterized VZV strains. Based on genotype A, D, B, and C reference strains, a dichotomous identification key (DIK) was developed and applied for VZV strains obtained from vesicle fluid and liquor samples originating from 42 patients suffering from varicella or zoster between 2003 and 2006. Sequencing of regions in ORFs 51, 52, 53, 56, 57, and 58 identified 18 single-nucleotide polymorphisms (SNPs), including two novel ones, SNP 89727 and SNP 92792 in ORF51 and ORF52, respectively. The DIK as well as phylogenetic analysis by Bayesian inference showed that 14 VZV strains belonged to genotype A, and 28 VZV strains were classified as genotype D. Neither Japanese (vaccine)-like B strains nor recombinant-like C strains were found within the samples from Germany. The novel genotyping scheme and the DIK were demonstrated to be practical and simple and allow the highly efficient replication of phylogenetic patterns in VZV initially derived from full-genome DNA sequence analyses. Therefore, this approach may allow us to draw a more comprehensive picture of wild-type VZV strains circulating in Germany and Central Europe by high-throughput procedures in the future.

Novel sst5-selective somatostatin dicarba-analogues: synthesis and conformation-affinity relationships

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33

In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcepsilonRI-activation, and enhances FcepsilonRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-kappaB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.

Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy

OBJECTIVE: The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS: Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS: Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (-20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS: Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Value of a novel Neisseria meningitidis--specific polymerase chain reaction assay in skin biopsy specimens as a diagnostic tool in chronic meningococcemia

BACKGROUND: Chronic meningococcemia (CM) is a diagnostic challenge. Skin lesions are frequent but in most cases nonspecific. Polymerase chain reaction (PCR)-based diagnosis has been validated in blood and cerebrospinal fluid for acute Neisseria meningitidis infection, in patients in whom routine microbiologic tests have failed to isolate the bacteria. In 2 patients with CM, we established the diagnosis by a newly developed PCR-based approach performed on skin biopsy specimens. OBSERVATIONS: Two patients presented with fever together with systemic and cutaneous manifestations suggestive of CM. Although findings from blood cultures remained negative, we were able to identify N meningitidis in the skin lesions by a newly developed PCR assay. In 1 patient, an N meningitidis strain of the same serogroup was also isolated from a throat swab specimen. Both patients rapidly improved after appropriate antibiotherapy. CONCLUSIONS: To our knowledge, we report the first cases of CM diagnosed by PCR testing on skin biopsy specimens. It is noteworthy that, although N meningitidis-specific PCR is highly sensitive in blood and cerebrospinal fluid in acute infections, our observations underscore the usefulness of PCR performed on skin lesions for the diagnosis of chronic N meningitidis infections. Whenever possible, this approach should be systematically employed in patients for whom N meningitidis infection cannot be confirmed by routine microbiologic investigations.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

Novel weapons: Invasive plant suppresses fungal mutualists in America but not in its native Europe

Why some invasive plant species transmogrify from weak competitors at home to strong competitors abroad remains one of the most elusive questions in ecology. Some evidence suggests that disproportionately high densities of some invaders are due to the release of biochemicals that are novel, and therefore harmful, to naive organisms in their new range. So far, such evidence has been restricted to the direct phytotoxic effects of plants on other plants. Here we found that one of North America's most aggressive invaders of undisturbed forest understories, Alliaria petiolata (garlic mustard) and a plant that inhibits mycorrhizal fungal mutualists of North American native plants, has far stronger inhibitory effects on mycorrhizas in invaded North American soils than on mycorrhizas in European soils where A. petiolata is native. This antifungal effect appears to be due to specific flavonoid fractions in A. petiolata extracts. Furthermore, we found that suppression of North American mycorrhizal fungi by A. petiolata corresponds with severe inhibition of North American plant species that rely on these fungi, whereas congeneric European plants are weakly affected. These results indicate that phytochemicals, benign to resistant mycorrhizal symbionts in the home range, may be lethal to naive native mutualists in the introduced range and indirectly suppress the plants that rely on them.

Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Discovery of novel CB2 receptor ligands by a pharmacophore-based virtual screening workflow

Cannabinoid receptor 2 (CB(2) receptor) ligands are potential candidates for the therapy of chronic pain, inflammatory disorders, atherosclerosis, and osteoporosis. We describe the development of pharmacophore models for CB(2) receptor ligands, as well as a pharmacophore-based virtual screening workflow, which resulted in 14 hits for experimental follow-up. Seven compounds were identified with K(i) values below 25 microM. The CB(2) receptor-selective pyridine tetrahydrocannabinol analogue 8 (K(i) = 1.78 microM) was identified as a CB(2) partial agonist. Acetamides 12 (K(i) = 1.35 microM) and 18 (K(i) = 2.1 microM) represent new scaffolds for CB(2) receptor-selective antagonists and inverse agonists, respectively. Overall, our pharmacophore-based workflow yielded three novel scaffolds for the chemical development of CB(2) receptor ligands.

Endothelial cell protection by dextran sulfate: a novel strategy to prevent acute vascular rejection in xenotransplantation

We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cell-mediated cytotoxicity towards porcine cells in vitro. We therefore hypothesized that DXS, combined with cyclosporine A (CyA), could prevent acute vascular rejection (AVR) in the hamster-to-rat cardiac xenotransplantation model. Untreated, CyA-only, and DXS-only treated rats rejected their grafts within 4-5 days. Of the hearts grafted into rats receiving DXS in combination with CyA, 28% survived more than 30 days. Deposition of anti-hamster antibodies and complement was detected in long-term surviving grafts. Combined with the expression of hemoxygenase 1 (HO-1) on graft EC, these results indicate that accommodation had occurred. Complement activity was normal in rat sera after DXS injection, and while systemic inhibition of the coagulation cascade was observed 1 h after DXS injection, it was absent after 24 h. Moreover, using a fluorescein-labeled DXS (DXS-Fluo) injected 1 day after surgery, we observed a specific binding of DXS-Fluo to the xenograft endothelium. In conclusion, we show here that DXS + CyA induces long-term xenograft survival and we provide evidence that DXS might act as a local EC protectant also in vivo.