Antifungal activities of SCY-078 (MK-3118) and standard antifungal agents against clinical non-Aspergillus mold isolates.

The limited armamentarium of active and oral antifungal drugs against emerging non-Aspergillus molds is of particular concern. Current antifungal agents and the new orally available beta-1,3-d-glucan synthase inhibitor SCY-078 were tested in vitro against 135 clinical non-Aspergillus mold isolates. Akin to echinocandins, SCY-078 showed no or poor activity against Mucoromycotina and Fusarium spp. However, SCY-078 was highly active against Paecilomyces variotii and was the only compound displaying some activity against notoriously panresistant Scedosporium prolificans.

The Aldosterone/Renin Ratio as a Diagnostic Tool for the Diagnosis of Primary Hypoaldosteronism in Newborns and Infants.

BACKGROUND/AIMS: Primary hypoaldosteronism is a rare inborn disorder with life-threatening symptoms in newborns and infants due to an aldosterone synthase defect. Diagnosis is often difficult as the plasma aldosterone concentration (PAC) can remain within the normal range and thus lead to misinterpretation and delayed initiation of life-saving therapy. We aimed to test the eligibility of the PAC/plasma renin concentration (PRC) ratio as a tool for the diagnosis of primary hypoaldosteronism in newborns and infants. Meth ods: Data of 9 patients aged 15 days to 12 months at the time of diagnosis were collected. The diagnosis of primary hypoaldosteronism was based on clinical and laboratory findings over a period of 12 years in 3 different centers in Switzerland. To enable a valid comparison, the values of PAC and PRC were correlated to reference methods. RESULTS: In 6 patients, the PAC/PRC ratio could be determined and showed constantly decreased values <1 (pmol/l)/(mU/l). In 2 patients, renin was noted as plasma renin activity (PRA). PAC/PRA ratios were also clearly decreased. The diagnosis was subsequently genetically confirmed in 8 patients. CONCLUSION: A PAC/PRC ratio <1 pmol/mU and a PAC/PRA ratio <28 (pmol/l)/(ng/ml × h) are reliable tools to identify primary hypoaldosteronism in newborns and infants and help to diagnose this life-threatening disease faster. © 2015 S. Karger AG, Basel.

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09.

OBJECTIVE: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced non-small-cell lung cancer are promising for further investigation. METHODS: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. RESULTS: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16 cycles. PFS at 6 months was 70%, median PFS was 14 months, and ORR was 70%. Biopsy at progression was safe and successful in 71% of the cases. CONCLUSIONS: Both combination therapies were promising for further studies. Biopsy at progression was feasible and will be part of future SAKK studies to investigate molecular mechanisms of resistance.

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max. L.)

Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.

Germination, senescence and pathogenic attack in soybean (Glycine max. L.): Identification of the cytosolic aconitase participating in the glyoxylate cycle

During our study of the glyoxylate cycle in soybean (Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between P-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the CI aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

BACKGROUND: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. RESULTS: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. CONCLUSIONS: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells.

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells.

Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO) is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS) for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2(+)-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue), might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in humans.

Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production.

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.

Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.