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Previous work has shown that tyrosine residues Y423, Y428, and Y432 are potential targets for nitration when tyrosine hydroxylase is exposed to peroxynitrite. For any given protein molecule, up to three nitration events involving these residues may occur. In an effort to determine whether this nitration is directed toward a particular tyrosine residue or randomly distributed among all three tyrosine residues, the goal of this study was to determine whether a singly nitrated molecule was always nitrated at a particular tyrosine residue or whether the initial nitration event was randomly distributed among all three tyrosine residues. Isolation of the singly nitrated peptide V410-E436, followed by subsequent cleavage at D425 and analysis of the resulting peptide fragments by matrix-assisted laser desorption ionization mass spectrometry and LC/MS/MS revealed that the first nitration event was randomly distributed among Y423, Y428, and Y432.

SUMMARYDamp buildings often have a moldy smell or obvious mold growth; some molds are human pathogens. This has caused concern regarding health effects of moldy indoor environments and has resulted in many studies of moisture- and mold-damaged buildings. Recently, there have been reports of severe illness as a result of indoor mold exposure, particularly due to Stachybotrys chartarum. While many authors describe a direct relationship between fungal contamination and illness, close examination of the literature reveals a confusing picture. Here, we review the evidence regarding indoor mold exposure and mycotoxicosis, with an emphasis on S. chartarum. We also examine possible end-organ effects, including pulmonary, immunologic, neurologic, and oncologic disorders. We discuss the Cleveland infant idiopathic pulmonary hemorrhage reports in detail, since they provided important impetus for concerns about Stachybotrys. Some valid concerns exist regarding the relationship between indoor mold exposure and human disease. Review of the literature reveals certain fungus-disease associations in humans, including ergotism (Claviceps species), alimentary toxic aleukia (Fusarium), and liver disease (Aspergillys). While many papers suggest a similar relationship between Stachybotrys and human disease, the studies nearly uniformly suffer from significant methodological flaws, making their findings inconclusive. As a result, we have not found well-substantiated supportive evidence of serious illness due to Stachybotrys exposure in the contemporary environment. To address issues of indoor mold-related illness, there is an urgent need for studies using objective markers of illness, relevant animal models, proper epidemiologic techniques, and examination of confounding factors.

ABSTRACTBiofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candidabiofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicansand Candida parapsilosisbiofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candidabiofilms. We also explored effects of preincubation of C. albicanscells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candidabiofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candidaisolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candidabiofilms. Preincubation of C. albicanscells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P< 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candidabiofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

ABSTRACTLittle is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candidaspecies, particularly Candidaalbicansand C. parapsilosis, was evaluated by using a clinically relevant model of Candidabiofilm on medical devices. Candidabiofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicansisolates were seen, significant differences between invasive and noninvasive isolates (P< 0.001) were noted. C. albicansisolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalisisolates, as determined by DW assays (Pwas <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicansbiofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosisbiofilms had less volume than C. albicansbiofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candidabiofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 μg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicansproduces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Tryptophan hydroxylase [tryptophan 5-monooxygenase, L-tryptophan,tetrahydropterin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] is activated by phosphorylating conditions (ATP-Mg2+) in a calcium-dependent, cyclic AMP-independent manner. Addition to the phosphorylation reaction of certain antipsychotic drugs that bind to calmodulin, the heat-stable calcium-binding protein, prevents the activation of tryptophan hydroxylase by ATP-Mg2+ in a concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Calmodulin-free tryptophan hydroxylase prepared by affinity chromatography on fluphenazine-Sepharose is not activated by ATP-Mg2+ whereas addition of calmodulin to calmodulin-free enzyme restores the responsiveness of the hydroxylase to ATP-MG2+ only in the presence of Ca2+. These results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.

The effects of L-tryptophan on blood pressure and brain serotonin have been investigated in normotensive and spontaneously hypertensive rats. Doses of L-tryptophan from 25 to 100 mg/kg increased the blood pressure of normotensive rats by 10 to 15 mm Hg. The lower doses of L-tryptophan also increased blood pressure in hypertensive rats but, in contrast, the highest dose (100 mg/kg) lowered blood pressure by 30 to 35 mm Hg. Blood pressure effects of L-tryptophan were evident within 30 min of treatment and reached maximal response by 60 min. L-Tryptophan-induced changes in serotonin neurochemistry were correlated on a temporal basis with changes in blood pressure. However, the dose-response relationship between blood pressure changes and increases in brain serotonin and 5-hydroxyindoleacetic acid content produced by L-tryptophan was not related. The effects of L-tryptophan on blood pressure in normotensive and spontaneously hypertensive rats cannot be explained entirely by its effects on brain serotonin.

Pentazocine (10 mg/kg) and saline were used as discriminative stimuli in rats. After pentazocine administration, reinforcement could be obtained contingent on pressing one particular lever (left or right) in a two-lever operant chamber. Responding on the opposite lever was reinforced only after saline injections; a drug-lever combination was always held constant for each animal. Discriminated choice responding between pentazocine and saline was learned to an 80% correct criterion. Test injections of morphine produced dose-related responding on the pentazocine lever. Choice responding after 7.5 mg/kg of morphine was not significantly different from choice responding after 10 mg/kg of pentazocine. The discriminable ED50 values for both pentazocine and morphine were estimated from dose-response curves and when given in combination (pentazocine ED50 + morphine ED50), more drug-related responding occurred than occurred after either drug (ED50) alone. Naloxone produced saline-like responding but antagonized the stimulus properties of both pentazocine and morphine. However, the antagonism of the morphine cue by naloxone was significantly greater than the antagonism of the pentazocine cue. The results indicate that pentazocine and morphine are similar in at least one important respect suggesting that these drugs may share a common site of action.

Electrical stimulation (1--50 Hz, 100--500 muA, 0.3 msec pulse duration) of the dorsal or median raphe nucleus in anesthetized rats produces a transient increase in arterial blood pressure. Heart rate and respiratory rate do not appear to change systematically after stimulation. Transections rostral or caudal to the raphe nuclei or electrolytic lesions in the area of the nucleus linearis, pars caudalis abolishes the pressor response to stimulation. Depletion of brain serotonin with p-chlorophenylalanine significantly attenuates the pressor effect of both dorsal and median raphe stimulation. The p-chlorophenylalanine effect can be partially reversed by Ro4-4602, a peripherally acting decarboxylase inhibitor, plus 5-hydroxytryptophan. Fluoxetine, a specific serotonin uptake inhibitor, prolongs the duration of the raphe pressor response and slightly increases its magnitude. Injections of 2-bromolysergic acid diethylamide (BOL), a putative serotonin antagonist, into the anterior hypothalamus/preoptic area significantly attenuates the dorsal raphe pressor effect, whereas treatment of rats with the sympathetic postganglionic blocking agent bretylium prevents the stimulation-induced pressor effect. Taken together, these data suggest that stimulation of the ascending serotonergic neuronal system produces a phasic pressor effect which is mediated, at least in part, by synaptic serotonin.

(6R)-5,6,7,8-Tetrahydrobiopterin (BH4), which is synthesized intracellularly from GTP, caused a concentration-dependent increase in rat pheochromocytoma (PC12) cell proliferation when added exogenously. Incubation with sepiapterin, which is converted enzymatically to BH4 within cells, also increased PC12 cell proliferation and BH4 levels concomitantly. These sepiapterin effects were mediated by BH4 as inhibition of sepiapterin conversion to BH4 by a sepiapterin reductase inhibitor, N-acetyl-serotonin, blocked the increase in proliferation and the elevation of BH4 levels. 7,8-Dihydrobiopterin (BH2) also increased BH4 levels and PC12 cell proliferation, both of which were reversed by methotrexate, which blocks the conversion of BH2 to BH4 by dihydrofolate reductase. The BH4-induced increase in PC12 cell proliferation was not related to elevated catecholamine or nitric oxide synthesis as inhibitors of tyrosine hydroxylase or nitric oxide synthase did not reduce the BH4 effect. BH4 and its precursors did not alter intracellular cAMP levels, suggesting that this second messenger is not involved in the enhancement of PC12 cell proliferation by BH4. Sepiapterin and BH4 also enhanced the proliferation of SV40-transformed human fibroblasts and rat C6 glioma cells, indicating that the stimulatory effect of BH4 on cell proliferation is not restricted to PC12 cells.

Calmodulin was removed from brain cytosol by DEAE-52 chromatography or by affinity chromatography employing fluphenazine-Sepharose. The substrates phosphorylated by endogenous protein kinase after chromatography differed depending on the method used, and both chromatographic methods altered the phosphorylation pattern as compared to untreated cytosol. Cytosol, chromatographed on fluphenazine-Sepharose, retained most of the characteristics of untreated cytosol. Both calmodulin and phospholipids increased the phosphorylation of specific but separate brain cytosol proteins in a Ca2+-dependent manner. The effects of phospholipids could be mimicked by the detergent, sodium dodecyl sulfate, and the hydrophobic probe, 8-anilino-1-naphthalenesulfonate. Furthermore, the calmodulin-induced increase in phosphorylation, but not that produced by phospholipids, was blocked by 8-anilino-1-naphthalenesulfonate. These results suggest that the effects of phospholipids may not be due to the presence of a specific phospholipid-sensitive protein kinase in cytosol, but rather to a general interaction of hydrophobic probes with either specific substrate proteins or with the Ca2+-calmodulin-dependent protein kinase itself.

ABSTRACTColorimetric tetrazolium assays are used increasingly in studies of fungi, often in the absence of standardization or correlation with other methods. We examined species- and strain-related tetrazolium metabolism in Candida albicansand Candida parapsilosisby using XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} and WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphonyl)-2H-tetrazolium] and found marked variations. Also, significant signal was often missed in the absence of dimethyl sulfoxide extraction.