Complex multipathways alterations and oxidative stress are associated with Hailey-Hailey disease.

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene that encodes the Ca(2+)/Mn(2+)-adenosine triphosphatase ATP2C1 and HHD, we still have little understanding of how these mutations affect manifestations of the disease. OBJECTIVES: This study was designed to determine early signalling events that affect epithelial cell growth and differentiation during HHD development. METHODS: Expression of key regulatory signals important for maintaining skin homeostasis were evaluated by Western blot analysis and by reverse transcriptase-polymerase chain reaction in primary keratinocytes obtained from skin biopsies of patients with HHD. Reactive oxygen species accumulation in primary keratinocytes derived from lesional skin of patients with HHD was assessed by dihydrorhodamine 123 (DHR) assay. RESULTS: HHD-derived keratinocytes showed downregulation of both Notch1 and differential regulation of different p63 isoforms. Itch and p63 are co-expressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. We found that the Itch protein was significantly decreased in HHD-derived keratinocytes whereas the expression of its target, c-Jun, remained unaffected. We also found that HHD-derived keratinocytes undergo oxidative stress, which may explain both Notch1 and Itch downregulation. CONCLUSIONS: Our attempt to explore the molecular mechanism underlying HHD indicates a complex puzzle in which multi-hit combinations of altered signal pathways may explain the wide spectrum of defects in HHD.

Synthesis and NMR-driven conformational analysis of taxol analogues conformationally constrained on the C13 side chain.

Analogues of Taxol (paclitaxel) with the side chain conformationally restricted by insertion of a carbon linker between the 2'-carbon and the ortho-position of the 3'-phenyl ring were synthesized. Biological evaluation of these new taxoids showed that activity was dependent on the length of the linker and the configuration at C2' and C3'. Two analogues in the homo series, 9a and 24a, showed tubulin binding and cytotoxicity comparable to that of Taxol. NAMFIS (NMR analysis of molecular flexibility in solution) deconvolution of the averaged 2-D NMR spectra for 9a yields seven conformations. Within the latter set, the hydrophobically collapsed "nonpolar" and "polar" classes are represented by one conformation each with predicted populations of 12-15%. The five remaining conformers, however, are extended, two of which correspond to the T-conformation (47% of the total population). The latter superimpose well with the recently proposed T-Taxol binding conformer in beta-tubulin. The results provide evidence for the existence of two previously unrecognized structural features that support Taxol-like activity: (1) a reduced torsion angle between C2' and C3' and (2) an orthogonal arrangement of the mean plane through C1', C2' and the 2'-hydroxyl and the 3'-phenyl plane, the latter ring bisected by the former plane. By contrast, epimerization at 2',3' and homologation of the tether to CH2-CH2 were both detrimental for activity. The decreased activity of these analogues is apparently due to configurational and steric factors, respectively.

Two new tryptophan derivatives from the seed kernels of Entada rheedei: effects on cell viability and HIV infectivity.

Two new tryptophan derivatives, N-sulfonyl-L-tryptophan (tryptorheedei A) (1) and 3-(N-sulfonylindolyl)-D-lactic acid (tryptorheedei B) (2) together with the known 5-O-ß-D-glucopyranosyl-2-hydroxyphenylacetic acid (3), 1-O-methylglucopyranoside, entadamide A, homogentisic acid and 3-O-ß-D-glucopyranosyl-ß-sitosterol, were isolated from the seed kernels of Entada rheedei (Mimosaceae). Their structures were established using 1D and 2D NMR spectroscopy, mass spectrometry and by comparison with spectroscopic data reported in the literature. Compounds 1 and 2 showed no toxicity to TZM and Human PBMC cells. Both compounds 1 and 2 were found to promote early infection events in HIV, likely by inhibiting the enzyme indolamine 2,3-dioxygenase (IDO) and preventing tryptophan depletion. Inhibition of IDO acutely in HIV infection inhibits viral replication, but chronic activation of IDO leads to immune impairment in AIDS. IDO is also the gatekeeper enzyme for kynurenine metabolism, a pathway involved in serotonin and melatonin biosynthesis and the regulation of glutamate and dopamine levels in the brain. Therefore inhibition of IDO might explain both the reported medicinal and neuropsychiatric effects of E. rheedei.

HPLC quantification of coumarin in bastard balm (Melittis melissophyllum L., Lamiaceae).

Coumarin occurs in many plants used as flavoring and is known to possess hepatotoxic effects. Despite in the EFSA 'Compendium of botanicals containing toxic substances' coumarin is reported to be present in Melittis melissophyllum (bastard balm), a plant traditionally used as beverage in Italy and Serbia, to the best of our knowledge quantitative data has never been reported. Thus, the amount of coumarin in bastard balm leaves and its variation during the annual phenological cycle were determined. The subsp. melissophyllum resulted to contain high levels of coumarin (14,392 mg/kg), mainly in the early stages of the plant cycle, suggesting prudence in its use as beverage. Furthermore, coumarin was found to be useful as marker compound to differentiate the bastard balm subspecies occurring in Italy, since the subsp. albida contained a much lower content of this molecule (19-34 mg/kg).

Minor taxoids from Taxus wallichiana.

The needles of Taxus wallichiana afforded a new analogue of taxinine M [1a] and two derivatives of brevifoliol [2a, 3a]. The conformation of 3a was investigated by nmr spectroscopy with the aid of variable-temperature experiments and in situ reactions.

Immunorecognition of ring skeleton of taxanes by chicken egg yolk antibodies.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

Novel d-seco paclitaxel analogues: synthesis, biological evaluation, and model testing.

Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis involved the opening of the D-ring by Jones oxidation. Two of the compounds had been predicted to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological results identify a weakness in our predictive minireceptor model and suggest a corrective remedy in which additional amino acids are needed to accommodate ligand-protein steric effects around the oxetane ring. These changes to the model lead to correct predictions of the bioactivity. Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl substituent is as important as the oxetane in determining the A ring conformation.