Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

The design and characterization of receptor-selective APRIL variants.

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

The Selective Phosphodiesterase 4 Inhibitor Roflumilast and Phosphodiesterase 3/4 Inhibitor Pumafentrine Reduce Clinical Score and TNF Expression in Experimental Colitis in Mice.

OBJECTIVE: The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. METHODS: The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. RESULTS: Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. CONCLUSIONS: These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice.

Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription.

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARgamma and PPARbeta, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARgamma and PPARbeta and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARbeta-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions.

Analytical methods for the quantitative determination of selective serotonin reuptake inhibitors for therapeutic drug monitoring purposes in patients.

Five selective serotonin reuptake inhibitors (SSRIs) have been introduced recently: citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline. Although no therapeutic window has been defined for SSRIs, in contrast to tricyclic antidepressants, analytical methods for therapeutic drug monitoring of SSRIs are useful in several instances. SSRIs differ widely in their chemical structure and in their metabolism. The fact that some of them have N-demethylated metabolites, which are also SSRIs, requires that methods be available which allow therapeutic drug monitoring of the parent compounds and of these active metabolites. most procedures are based on prepurification of the SSRIs by liquid-liquid extraction before they are submitted to separation by chromatographic procedures (high-performance liquid chromatography, gas chromatography, thin layer chromatography) and detection by various detectors (UV, fluorescence, electrochemical detector, nitrogen-phosphorus detector, mass spectrometry). This literature review shows that most methods allow quantitative determination of SSRIs in plasma, in the lower ng/ml range, and that they are, therefore, suitable for therapeutic drug monitoring purposes of this category of drugs.

Cardiac Toxicity in Selective Serotonin Reuptake Inhibitor Users.

Several classes of recreational and prescription drugs have been associated with an increased risk of cardiovascular disease and the occurrence of arrhythmias, which may be involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart, which may be caused by selective serotonin reuptake inhibitor use and their possible role in the occurrence of sudden cardiac death. A total of 40 cases were included in the study and were divided evenly into 2 groups: 20 cases of patients treated with selective serotonin reuptake inhibitors and 20 cases of sudden deaths involving patients void of any drug treatment. The first group included 16 patients treated with citalopram and 4 with sertraline. Autopsies, histology, biochemistry, and toxicology were performed in all cases. Pathological changes in selective serotonin reuptake inhibitor users consisted of various degrees of interstitial and perivascular fibrosis as well as a small degree of perineural fibrosis within the myocardium of the left ventricle. Within the limits of the small number of investigated cases, the results of this study seem to confirm former observations on this topic, suggesting that selective serotonin reuptake inhibitors may play a potential, causative role in the pathogenesis of sudden deaths in chronic users even at therapeutic concentrations.