Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.     

Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models.

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF beta were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-gamma-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-gamma treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-beta synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-beta, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.     

Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granulomatous inflammation.

The role of RANTES in Th1 and Th2 cell-mediated immune responses has been enigmatic. To approach this question, we analyzed RANTES expression and function in murine models of types 1 and 2 cell-mediated pulmonary granulomas elicited with Mycobacterium bovis or Schistosoma mansoni egg Ag-coated beads, respectively. Compared with type 2, type 1 lesions had up to 4-fold greater RANTES protein and mRNA production. Type 1 draining lymph nodes also produced up to 7-fold higher levels of RANTES. Anti-RANTES Ab treatments had opposite effects, decreasing type 1 lesion area by 25% and augmenting type 2 lesions by 50%. The latter was associated with increased IL-4, IL-5, IL-10, and IL-13 production by lymph nodes. Infusion of rRANTES (1 mg/kg/day) did not affect type 1 lesions, but reduced type 2 lesion area by 27% and eosinophils by 40%. Lymph node cultures from RANTES-treated mice had augmented type 1 and impaired type 2 responses. In vitro, RANTES caused selective, dose-related inhibition of IL-4 that was largely dependent on CCR1 receptors. In conclusion, RANTES plays different roles in types 1 and 2 granuloma formation, promoting the former and mediating cross-regulatory inhibition of the latter. Moreover, RANTES may have therapeutic potential in the treatment of established type 2 hypersensitivity.     

Diving deeper into the underlying white shark behaviors at Guadalupe Island,Mexico

1. Fine‐scale movement patterns are driven by both biotic (hunting, physiological needs) and abiotic (environmental conditions) factors. The energy balance governs all movement‐related strategic decisions.
2. Marine environments can be better understood by considering the vertical component. From 24 acoustic trackings of 10 white sharks in Guadalupe Island, this study linked, for the first time, horizontal and vertical movement data and inferred six different behavioral states along with movement states, through the use of hidden Markov models, which allowed to draw a comprehensive picture of white shark behavior.
3. Traveling was the most frequent state of behavior for white sharks, carried out mainly at night and twilight. In contrast, area‐restricted searching was the least used, occurring primarily in daylight hours.
4. Time of day, distance to shore, total shark length, and, to a lesser extent, tide phase affected behavioral states. Chumming activity reversed, in the short term and in a nonpermanent way, the behavioral pattern to a general diel vertical pattern.

     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.     

Toll-like receptor 4 mediates vascular remodeling in hyperhomocysteinemia

Nephrotic syndrome (NS) is a kidney disease predominantly present in children with idiopathic condition; final stage of the disease progresses into end-stage renal disease. Generally, NS is treated using standard steroid therapy, however; most of the children are steroid sensitive and about 15–20% are non-responders (SRNS). Non-responsiveness of these children would be a risk with the possibility of mutational changes in podocyte genes (NPHS1, NPHS2, WT1, PLCE1). The mutation in podocyte genes is associated with SRNS. NPHS1, NPHS2, and WT1 genes are identified/directly linked to SRNS. The present study is a surveillance on the mutation analysis of WT1 (exons 8 and 9) and NPHS2 (exons 1–8) gene in SRNS followed by clinical management. In the present study, we analyzed these two genes in a total of 117 SRNS (73 boys and 44 girls) children. A total of five mutations were detected in six children. First, WT1 mutation was detected at 9th intron-IVS 9 + 4C > T position in one SRNS female patient. This WT1 mutation was identified in a girl having Frasier Syndrome (FS) with focal segmental glomerulosclerosis and a complete sex reversal found through molecular and karyological screening. In NPHS2, missense mutations of P20L (in two children), P316S, and p.R229Q, and a frame shift mutation of 42delG were detected. Thus, applying molecular investigation helped us to decide on treatment plan of SRNS patients, mainly to avoid unnecessary immunosuppressive treatment.