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The cell-wall constituent, melanin, is a virulence factor for pathogenic fungi, but its structural and mechanistic role is not clearly understood. As intermediates in melanin formation are cross-linking agents, we wondered whether melanized cell walls might be more highly cross-linked and less porous than non-melanized cell walls. The fungal pathogen Cryptococcus neoformans makes melanin only in the presence of exogenous catechols; we cultivated it with and without 1 mmol/l dopamine. We prepared mechanically intact melanized and non-melanized cell walls by boiling cells in 10% sodium dodecyl sulfate; electron microscopy showed disruption of cytoplasm. We poured the resulting spheres into columns and studied the elution behavior of graded dextrans. High-molecular-weight dextrans eluted earlier than low-molecular-weight dextrans, which, in turn, eluted before glucose, behavior characteristic of size-exclusion chromatography. We calculated the thresholds above which the polymers were totally excluded from the cell walls. Melanized cells exhibited a threshold of molecular weight 30 600, non-melanized, 270 000 (P <0.01). The corresponding Einstein-Stokes radii are 4.0 and 10.6 nm, respectively; these represent the calculated largest pore sizes for each condition. We conclude that melanized cell walls are considerably less porous than non-melanized cell walls.

Randomly obtained, constitutive plasma membrane ferric reductase/ferrous uptake mutants of Cryptococcus neoformans were mapped to four distinct loci by meiotic analysis. One of those loci, FRR1 , was previously found homologous to MRS3 and MRS4 of Saccharomyces cerevisiae , which determine proteins involved in mitochondrial transport of iron. We were able to complement, clone, sequence and thereby identify two of the three remaining constitutive uptake loci. FRR3 was found to be homologous to ISU1 and ISU2 of S. cerevisiae, which form mitochondrial iron-sulfur complexes; FRR4 was found to be homologous to YFH1, the yeast frataxin homologue, which also participates in iron-sulfur cluster biogenesis. Because of the constitutive iron uptake seen in these mutants, mitochondria appear to have a central role in the cellular iron economy; moreover, as judged by our mutational statistics, the genetic machinery for mitochondrial iron accumulation may be more complex than that of the cytoplasm.

Cryptococcus neoformans is subject to oxidative attack by host immune cells; consequently, oxidant-resistant mechanisms may be important in pathogenesis. Mutations at the OXY2 locus confer decreased laccase and increased sensitivity to hyperbaric oxygen in the background of the oxyl mutation, but, alone, do not confer sensitivity to oxidants. Because metal deficiency can potentiate or ameliorate sensitivity to oxidants, and because the melanin-synthesizing laccase contains copper, we investigated copper acquisition in an oxy2 mutant. We found that its external Cu/Fe reductase activity was lower than that of wild type, and although copper deprivation induced the reductase in the wild type, it did not do so in oxy2. Oxy2 is sensitive to copper chelation but resistant to high copper, suggesting that copper transport is decreased. The strain expresses large amounts of alternate oxidase in response to Cu-chelation, perhaps in response to defective, Cu-deprived cytochrome oxidase, and is resistant to the oxidant, plumbagin, under this condition, perhaps due to the high alternate oxidase. These phenotypes are similar to those of the mac1- mutant of Saccharomyces cerevisiae and the melanin-deficient grisea mutant of Podospora anserina, in which homologous transcriptional activators for the reductase and copper transporter genes are mutated. They constitute physiologic evidence that oxy2 is mutated in a homologous copper-related transcriptional activator of C. neoformans.

Cryptococcus neoformans secretes 3-hydroxyanthranilate (3HAA), but the utility is unknown. Exogenous 3HAA promoted growth of cultures starved for iron with transferrin, presumably by releasing Fe(III) reductively. Exogenous 3HAA protected C. neoformans from strong oxidants, suggesting a role in resistance to killing by immune cells. 3HAA represents an endogenous laccase substrate, in that crude laccase preparations convert 3HAA to cinnabarinic acid, whereas 3HAA concentrations are higher in Lac- mutants. We isolated hypersecreting mutants as highly fluorescent clones. Because C. neoformans has been isolated from rotting wood, we looked for a role in degradation of lignin. Using cyclic voltammetry, we found no electrochemical evidence that organic oxidation products of 3HAA are capable of oxidizing lignin. We found neither cellulose dehydrogenase nor lignin peroxidase enzymic activity, nor did C. neoformans grow on cellulose as carbon source. We found no evidence for production of Fenton reagent by cultures, even in the presence of transition metal ions or of those and 3HAA. The biological utility of 3HAA may be related to its functions as reducing agent and, conceivably, as laccase substrate. It does not appear to attack wood, nor does C. neoformans appear to have a mechanism to rot wood.

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.

Cryptococcus neoformans is a pathogenic basidiomycete that causes meningitis in immunocompromised patients. In this paper, we demonstrate that a previously described oxidant-sensitive mutant, oxy1, has constitutive ferric reductase and iron uptake, similar to a ferric reductase regulatory mutant, frr1. Through meiotic genetic analysis, we show that the two mutations are allelic. By complementation of frr1 with a genomic library, we isolated a gene, MRS3/4. The encoded protein is a putative solute transporter of the inner mitochondrial membrane. Disruption of this gene led to high ferric reductase, iron uptake and iron content, as well as increased sensitivity to hydrogen peroxide and slow growth in low iron medium. The disrupted gene is allelic with oxy1 and frr1. We sequenced the oxy1 and frr1 alleles of MRS3/4 and found that the frr1 mutation results in a premature stop codon, while the oxy1 mutation results in the substitution of a highly conserved glutamate residue with lysine. The Mrs3/4 protein appears to be involved in mitochondrial iron transport in eukaryotes. Resistance to strong oxidants requires stringent control of iron metabolism.

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.

Melanins represent virulence factors for several pathogenic fungi; the number of examples is growing. Thus, albino mutants of several genera (in one case, mutated precisely in the melanizing enzyme) exhibit decreased virulence in mice. We consider the phenomenon in relation to known chemical properties of melanin, beginning with biosynthesis from ortho-hydroquinone precursors which, when oxidized enzymatically to quinones, polymerize spontaneously to melanin. It follows that melanizing intermediates are cross-linking reagents; melanization stabilizes the external cell wall against hydrolysis and is thought to determine semipermeability in the osmotic ram (the appressorium) of certain plant pathogens. Polymeric melanins undergo reversible oxidation-reduction reactions between cell wall-penetrating quinone and hydroquinone oxidation states and thus represent polymeric redox buffers; using strong oxidants, it is possible to titrate the melanin on living cells and thereby demonstrate protection conferred by melanin in several species. The amount of buffering per cell approximately neutralizes the amount of oxidant generated by a single macrophage. Moreover, the intermediate oxidation state, the semiquinone, is a very stable free radical and is thought to trap unpaired electrons. We have suggested that the oxidation state of external melanin may be regulated by external Fe(II). An independent hypothesis holds that in Cryptococcus neoformans, an important function of the melanizing enzyme (apart from melanization) is the oxidation of Fe(II) to Fe(III), thereby forestalling generation of the harmful hydroxyl radical from H(2)O(2). Thus, problems in fungal pathogenesis have led to evolving hypotheses regarding melanin functioning.

Cryptococcus neoformans is a pathogenic yeast that causes meningitis in immunocompromised patients. Because iron acquisition is critical for growth of a pathogen in a host, we studied the regulation of the ferric reductase and ferrous uptake system of this organism. We isolated 18 mutants, representing four independent loci, with dysregulated ferric reductase. The mutant strains had >10-fold higher than wild-type WT reductase activity in the presence of iron. Two of the strains also had >7-fold higher than WT iron uptake in the presence of iron but were not markedly iron sensitive. Both were sensitive to the oxidative stresses associated with superoxide and hydrogen peroxide. One strain exhibited only 23% of the WT level of iron uptake in the absence of iron and grew poorly without iron supplementation of the medium, phenotypes consistent with an iron transport deficiency; it was sensitive to superoxide but not to hydrogen peroxide. The fourth strain had high reductase activity but normal iron uptake; it was not very sensitive to oxidative stress. We also demonstrated that the ferric reductase was regulated by copper and could act as a cupric reductase. Sensitivity to oxidants may be related to iron acquisition by a variety of mechanisms and may model the interaction of the yeast with the immune system.

Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using 55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 microM, a plateau between 1 and 25 microM, and a second steep rise above 25 microM, consistent with high- and low-affinity uptake systems. A Km for high-affinity uptake was estimated to be 0.6 microM Fe(II); 1 microM was used for standardized uptake assays. At this concentration, the uptake rate was 110 +/- 3 pmol/10(6) cells/h. Iron repletion (15 microM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0 degreesC or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.

Melanin is a fungal extracellular redox buffer which, in principle, can neutralize antimicrobial oxidants generated by immunologic effector cells, but its source of reducing equivalents is not known. We wondered whether Fe(II) generated by the external ferric reductase of fungi might have the physiologic function of reducing fungal melanin and thereby promoting pathogenesis. We observed that exposure of a melanin film electrode to reductants decreased the open-circuit potential (OCP) and reduced the area of a cyclic voltammetric reduction wave whereas exposure to oxidants produced the opposite effects. Exposure to 10, 100, 1,000 or 10,000 microM Fe(II) decreased the OCP of melanin by 0.015, 0.038, 0.100, and 0.120 V, respectively, relative to a silver-silver chloride standard, and decreased the area of the cyclic voltammetric reduction wave by 27, 35, 50, and 83%, respectively. Moreover, exposure to Fe(II) increased the buffering capacity by 44%, while exposure to millimolar dithionite did not increase the buffering capacity. The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffering. Light absorption by melanin suspensions was decreased 14% by treatment with Fe(II), consistent with reduction of melanin. Light absorption by suspensions of melanized Cryptococcus neoformans was decreased 1.3% by treatment with Fe(II) (P < 0.05). Cultures of C. neoformans generated between 2 and 160 microM Fe(II) in culture supernatant, depending upon the strain and the conditions [the higher values were achieved by a constitutive ferric reductase mutant in high concentrations of Fe(III)]. We infer that Fe(II) can reduce melanin under physiologic conditions; moreover, it binds to melanin and cooperatively increases redox buffering. The data support a model for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular Fe(II) maintain the reducing capacity of the extracellular redox buffer.

The pathogenic yeast Cryptococcus neoformans must reduce Fe(III) to Fe(II) prior to uptake. We investigated mechanisms of reduction using the chromogenic ferrous chelator bathophenanthroline disulfonate. Iron-depleted cells reduced 57 nmol of Fe(III) per 10(6) cells per h, while iron-replete cells reduced only 8 nmol of Fe(III). Exponential-phase cells reduced the most and stationary-phase cells reduced the least Fe(III), independent of iron status. Supernatants from iron-depleted cells reduced up to 2 nmol of Fe(III) per 10(6) cells per h, while supernatants from iron-replete cells reduced 0.5 nmol of Fe(III), implying regulation of the secreted reductant(s). One such reductant is 3-hydroxyanthranilic acid (3HAA), which was found at concentrations up to 29 microM in iron-depleted cultures but <2 microM in cultures supplemented with iron. Moreover, when washed and resuspended in low iron medium, iron-depleted cells secreted 20.4 microM 3HAA, while iron-replete cells secreted only 4.5 microM 3HAA. Each mole of 3HAA reduced 3 mol of Fe(III), and increasing 3HAA concentrations correlated with increasing reducing activity of supernatants; however, 3HAA accounted for only half of the supernatant's reducing activity, indicating the presence of additional reductants. Finally, we found that melanized stationary-phase cells reduced 2 nmol of Fe(III) per 10(6) cells per h--16 times the rate of nonmelanized cells--suggesting that this redox polymer participates in reduction of Fe(III).

We examined the regulation of two fungal virulence factors, phenoloxidase and capsular polysaccharide, in an ex-type strain of the fungal pathogen, Cryptococcus neoformans. Both were made during the stationary phase of cultural growth. Exogenous iron increased phenoloxidase activity three-fold (from 8.7 to 27.7 units mg-1, P < 0.05) but decreased capsular polysaccharide three-fold (from 9.0 to 3.4% packed cell volume, P < 0.01). A temperature shift from 25 to 37 degrees C decreased phenoloxidase activity three-fold (from 60.6 to 23.7 units mg-1, P < 0.01) but not capsular polysaccharide (8.5 to 6.7% packed cell volume, P not significant). Thus, cryptococcal virulence factors are not regulated coordinately. Moreover, although the phenoloxidase synthesizes an antioxidant, melanin, the enzyme is not induced by the oxidant, hydrogen peroxide, or by a combination of hydrogen peroxide and solubilized ferric ion. As cryptococcal melanin is cheaply made from exogenous catechols, perhaps C. neoformans does not need to regulate the phenoloxidase strongly but, rather, can afford to synthesize the phenoloxidase at a moderate rate whenever it finds its growth limited.

Cryptococcus neoformans, an opportunistic pathogen, is the fourth leading cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure of glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was investigated by chemical analysis and 2D NMR spectroscopy. The assignment of 1H and 13C chemical shifts for the O-deacetylated polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enhanced HSQC spectra. The sequence and linkage positions of glycosyl residues were determined by NOESY and ROESY spectra. Two repeating polysaccharide components were identified as having the following structures in approximately equal proportions: [formula: see text] It is not known if these repeating units comprise a single or two separate polymer chains. Pentasaccharide 2 has been known to be the major GXM polymer of B-3501 and other serotype D isolates. Hexasaccharide 1 is identified for the first time although it has subsequently been identified in other C. neoformans isolates. The presence of 1 in the GXM of Cap70 is consistent with the extra xylose found relative to that in isolate B-3501. The mannose:xylose:glucuronic acid:O-acetyl molar ratio of Cap70 GXM is 3.00:1.73:0.78:1.75, while the same ratio for B-3501 and other serotype D isolates is approximately 3.00:1.00:0.80:1.75. Methylation analysis confirmed that the GXM of Cap70 contains unsubstituted, monosubstituted (2-linked), and disubstituted (2- and 4-linked) mannose in a ratio of 0.87:1.75:0.38. Dot blot immunoassay indicates that Cap70 is a serotype D isolate like its parent strain.

1,8-Dihydroxynaphthalene melanin in Wangiella dermatitidis and Alternaria alternata was titrated in vivo with the oxidants permanganate, hypochlorite, and H2O2. Melanized strains neutralized more oxidant and withstood higher concentrations of permanganate and hypochlorite than albino strains did. H2O2 killing required 1,000-fold higher concentrations, and melanin did not protect W. dermatitidis against H2O2.

Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization.

Since melanin is considered a virulence factor in Cryptococcus neoformans, its suppression at 37 degrees C has been perplexing. We now show an opposite thermal regulation of superoxide dismutase (SOD), consistent with a compensatory mechanism. Moreover, we demonstrate normal SOD and catalase levels in albino, oxygen-sensitive mutants. These results suggest that melanin is an antioxidant factor comparable in importance to SOD.

The polysaccharide capsule is a virulence factor in the opportunistic yeast pathogen, Cryptococcus neoformans. We describe a collection of strains which were isolated or constructed to exhibit defined capsular phenotypes. The collection includes strains with wild-type, acapsular and hypercapsular traits.

Polyphenols have been implicated in the virulence and oxidant resistance of Cryptococcus neoformans. Although monomeric polyphenols did not protect against the prooxidant, plumbagin, polymeric dopamine-melanin conferred resistance both to hypochlorite and to permanganate. The physiologic antioxidant capacity conferred by melanin was found to be 21.3 x 10(-15) mole-equivalents per cell, a value which approximates oxidant production by stimulated macrophages.

Iron is tightly controlled in mammalian tissues and regulates virulence factors in various pathogenic organisms. The influence of Fe availability upon production of cryptococcal capsular polysaccharide was studied. Polysaccharide, measured as cell-bound glucuronyl residues, increased more than threefold as available Fe in the culture medium was varied from repletion to tight sequestration and depletion in five incremental steps. Since physiologic CO2 concentration may serve as stimulus for cryptococcal polysaccharide synthesis, the combined effect of Fe availability and CO2 on encapsulation was studied. Addition of dissolved, loosely chelated Fe moderated the effect of CO2. Tight chelation of dissolved Fe potentiated the CO2 effect. Tissue from infected mice showed heavily encapsulated organisms, consistent with results with physiologic CO2 concentration and Fe deprivation. In conclusion, cryptococcal polysaccharide synthesis is increased by limitation of ferric iron availability to the cell and by dissolved CO2, and the two effects are additive.

A particulate enzyme preparation from Cryptococcus neoformans transferred the mannosyl residue from GDP-mannose to an acceptor consisting of a commercial preparation of methyl 3-O-alpha-mannopyranosyl-alpha-mannopyranoside (containing 10% 2-O-alpha-mannopyranosyl-alpha-mannopyranoside). The configuration of the new bond was alpha by its susceptibility to alpha-mannosidase; the amount of product was dependent on the concentration of enzyme, of GDP-mannose, and of acceptor. The optimal temperature and pH were 37 degrees C and 7.0, respectively. Manganous ion was required for activity and acetyl coenzyme A was stimulatory. Studies suggested that dolichyl phosphate intermediates were not involved in this mannose transfer. The fact that none of the several acapsular mutants tested were deficient in this mannosyltransferase suggested that this enzyme was not involved in synthesis of backbone mannan linkages in capsular polysaccharide. NMR analysis of the methylmannotriose product showed only alpha(1-->2) linkages between sugar moieties. This mannosyltransferase evidently extends alpha(1-->2) mannan by adding another alpha(1-->2)-linked mannosyl residue. Its activity is appropriate for a role in synthesis of "high mannose" oligosaccharide moieties of glycoproteins.

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH2-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.

Phenol oxidase enzymes, linked to virulence in Cryptococcus neoformans, were prepared from broken cells. More enzyme activity was found in the ultracentrifugation supernatant; less was found in the membrane fraction. Phenol oxidases were located in acrylamide gel electropherograms by activity staining with L-dihydroxyphenylalanine (DOPA). Mobility differences between soluble and solubilized membrane-bound phenol oxidases were not found. Comparison of enzymes produced at 25 and 37 degrees C revealed that the enzyme had lower activity and lower mobility at 37 degrees C. The mobility of 25 degrees C phenol oxidases from strains of C. neoformans var. gattii was lower than that of those from C. neoformans var. neoformans. Half of the phenol oxidase produced at 25 degrees C was bound by concanavalin A, while that produced at 37 degrees C was not bound. However, glucose starvation of cultures at 25 degrees C overnight resulted in increased amounts of enzyme which did not bind to concanavalin A. A given strain of C. neoformans produces different species of phenol oxidase under different culture conditions.

A comprehensive review of our series of surgical perilymphatic fistula (PLF) repairs, as well as a review of published results from other otologists, suggested an unacceptably high rate of postoperative PLF recurrence. Some recurrences were related to specific events (i.e., coughing, strenuous activity, Valsalva-type maneuvers). However many cases had no apparent cause. Rather, the patients' symptoms recurred spontaneously, and at reoperation the graft was seen to have not "taken," suggesting graft failure rather than "patient failure." After a critical evaluation of current PLF surgical procedures and state-of-the-art concepts of wound healing, we developed a new surgical technique for PLF closure. Combining the use of laser graft-site preparation, an autologous fibrin glue "buttress," and a program of postoperative activity restriction, the new procedure allowed us to achieve statistically significant improvements in graft retention and surgical outcome, with recurrences dropping from 27 percent to 8 percent. In addition, complete resolution or significant symptomatic improvement occurred in 89 percent of patients with vertigo and/or dizziness and in 84 percent with disequilibrium. We conclude that this new surgical technique is an important addition to the otologic surgeon's arsenal for PLF management.

We studied the effects of iron chelators and of a thallium salt on growth of Cryptococcus neoformans in defined medium. An oxidant-sensitive mutant strain was found to require exogenous ferric iron for growth. Using this strain, we found that the synthetic iron chelator, N-hydroxyethylenediamine triacetate (HEDTA), in several saturation states, stimulated growth as well as the comparably saturated siderophore deferoxamine. This non-specific result makes the existence of a cryptococcal ferrihydroxamate receptor doubtful. The catechols, caffeic acid, L-3, 4-dihydroxyphenylalanine, epinephrine, gallic acid, 3-hydroxytyramine (dopamine) and norepinephrine, were tested for growth stimulation in iron deprivation, under conditions in which deferoxamine was stimulatory. Catechols were found to be either neutral or inhibitory. The ferrous iron chelator, bathophenanthroline disulfonate (BPDS), inhibited growth strongly in the absence of exogenous iron, suggesting that ferric ion must be reduced before it can be internalized. Direct evidence of extracellular reduction was provided by accumulation of red-coloured ferrous-BPDS complex. The inhibition caused by BPDS was relieved by ferric HEDTA, even in the presence of 10-fold increased BPDS, suggesting a second, low-affinity, non-reductive iron uptake pathway. This inference was further supported by the observation that toxicity of the non-reducible ferric analogue, thallium (III), is relieved by iron repletion.

A wide range of recurrence rates (21% to 47%) for perilymph fistula repairs have been reported in the otology literature. An improved surgical technique developed at the Portland (Ore) Good Samaritan Hospital and Medical Center Neurotology Department was used to repair perilymph fistulas in 58 patients from October 1986 to October 1988. Our recurrence rate was reduced from 27% in a 1982-1985 study to 8% in our study. At 1 year postoperatively, improvements in disequillibrium, dizziness, and vertigo were comparable with results of older surgical techniques. Functional outcomes were also good: 83% of patients returned to normal activities of daily living, and 71% also returned to school or resumed gainful employment outside the home.

Melanin formation at 37 degrees C has been proposed as a virulence factor in Cryptococcus neoformans. However, whereas catecholamine uptake is maintained at this temperature, phenoloxidase, which catalyses the oxidation of catecholamine to melanin, is severely decreased in most wild type strains cultivated at 37 degrees C.

Oxygen sensitivity mutations of Cryptococcus neoformans were mapped to three genetic loci. Three oxygen-sensitive mutants had mutations that appeared allelic and exhibited albinism tightly linked to oxygen sensitivity; these three and a fourth exhibited defects in catechol uptake and catechol oxidation to melanin. Catecholamine metabolism appears to protect C. neoformans from oxidants.

The capsular polysaccharide of Cryptococcus neoformans consists of an O-acetylated mannan backbone with xylosyl and glucuronyl substituents. We have studied two enzymes in the biosynthetic pathway of this polysaccharide. A particulate enzyme preparation contained xylosyltransferase and glucuronyltransferase activities with pH optima of 7.5 and less than 6.5, and optimum incubation temperatures of 25 degrees and 37 degrees C respectively. O-acetylated mannan served as an acceptor for glucuronyl residues but xylomannan did not. Glucuronomannan served as an acceptor for xylosyl residues but acetylated mannan did not. These observations suggest that glucuronate is added before xylose and after acetate. This hypothesis was supported by studies of lipid-linked sugars in which a microsomal preparation was incubated with various combinations of nucleotide sugars. With GDP-mannose, UDP-glucuronate and UDP-[14C]xylose; GDP-mannose, UDP-[14C]glucuronate and UDP-xylose; or GDP-mannose and UDP-[14C]glucuronate, no evidence for a lipid-linked monosaccharide was obtained. However, lipid-linked xylose was detected only if GDP-mannose and UDP-[14C]xylose were provided, a finding consistent with a pool of lipid-linked xylose which is only transferred following glucuronylation. The order of addition inferred is mannosyl, acetyl, glucuronyl and xylosyl.

Between 1966 and 1977, 55 patients at Barnes Hospital were proven to have manifestations of histoplasmosis. Five patients had acute pulmonary histoplasmosis, four of whom demonstrated unusually severe disease. Disseminated histoplasmosis was documented in 19 cases. Four of these patients demonstrated dissemination during a severe and protracted but self-limited illness. The other 15 patients had progressive disease. Most of these patients were immune compromised and presented with fever of unknown origin. Therapy most benefited those who completed a course of greater than or equal to 1.5 gm of amphotericin B. Chronic pulmonary histoplasmosis was documented in 14 individuals. Large (> 1.5 cm), solitary pulmonary coin lesions, excised from 14 patients, were found to consist of exuberant granulomas containing yeast. Organisms were recovered from pleural effusions of two patients and demonstrated histologically in the tissue of one individual who presented with fibrosing mediastinitis.

There are few quantitative guidelines for the interpretation of serologic tests for histoplasmosis. Furthermore, guidelines that have been proposed are not accepted generally. This report reviews experiences with complement fixation and latex agglutination tests performed in a large acute-care hospital in an endemic area, and depicts the relationship between the proportion of false-positive reactions and the titer at which the serologic tests were reactive. We noted a considerable number of false-positive reactions for these widely-used tests. Use of a battery of standard tests for histoplasmosis resulted in a 90% decrease in false-positive reactions. Reliance upon multiple positive results in such a battery of tests would result in failure to detect 50% of cases of disseminated histoplasmosis, but would eliminate most false positives.

UDP glucuronate decarboxylase activity was comparable in encapsulated and non-encapsulated strains of Cryptococcus neoformans, required NAD (K(a) = 0.2 mM), and was inhibited by NADH (K(i) = 0.1 mM) and UDP xylose.

A pleitropic ethionine-resistant mutant of Neurospora contains an S-adenosylmethionine synthetase that is labile to heat and dialysis but exhibits normal kinetics for methionine and ethionine.

In the sexual phase of Cryptococcus neoformans four post-meiotic nuclei give rise to the nuclei of four long chains of basidiospores. If there is fixed relationship between one post-meiotic nucleus and one chain the result should be genetic homogeneity within single chains; random composition of chains would represent the opposite case. We tested for homogeneity in single chains from crosses of niacin-auxotrophs to the wild type by dissecting single chains of basidiospores from mature fruiting bodies. Out of 14 chains, 11 were homogeneous; the probability of obtaining this result by random processes was less than 0.01. These results suggest that mitotic products of a particular post-meiotic nucleus may be channeled into a particular basidiospore chain.

The polysaccharide capsule is a characteristic virulence factor in the yeast-pathogen, Cryptococcus neoformans. Growth in hypertonic growth media results in yeast cells with visibly smaller capsules. We investigated this suppression quantitatively, using a chemical assay for cell-bound and dissolved capsular polysaccharide. Molar NaCl suppressed production of cell-bound polysaccharide by a factor of 2.5- to 5-fold. The possibility of salt-induced physico-chemical contraction of capsular gel was tested by dialysis of fixed cells from hypotonic medium against medium containing 1 M NaCl and against the original medium again, while capsular thickness, packed cell volume and cell-bound polysaccharide were followed. We detected a physical contraction of gel following dialysis against medium containing 1 M NaCl. Mutants which gave mucoid colonies on hypertonic agar were isolated. One of these gave twice as much polysaccharide as the wild type when cultivated in medium containing 1 M NaCl. The hypercapsular trait was passed through serial outcrosses to the wild type and segregated as a chromosomal gene. This mutant may represent a gene which regulates production of capsular polysaccharide.

A mating between niacin and pantothenate auxotrophs of Cryptococcus neoformans gave a few prototrophic progeny that were self-fertile. These were uninuclear but contained twice as much DNA as the parental strains. Segregation of nutritional markers was observed upon sporulation. We conclude that these self-fertile strains are diploids.

Seven capsule-negative mutants of Cryptococcus neoformans were isolated. All mutations were linked (maximum map distance, 38 U); two mutations were found to be allelic.

The clinical presentation and autopsy findings in the case of a patient with acute myelomonocytic leukemia and trichinosis are described. Cellular immunity is an important host defense against nematode infections such as strongyloidiasis. It is therefore probable that the severe trichinosis seen in this immunosuppressed patient was more than coincidental. Due to concomitant failure of humoral immunity, serodiagnosis of trichinosis would have been impossible. The only means of making the diagnosis antemortem would have been muscle biopsy.

A cytologic method is described for the identification of trophoblastic epithelium in products of first-trimester abortion. In material from 50 consecutive induced first-trimester abortions, trophoblast was identified cytologically in 46 cellular samples (93.9%). In addition, nucleated fetal erythrocytes were found in 26 cellular samples (53%). There were no false positives, and three false negatives occurred. One negative examination, confirmed by histologic study, led to procedures that diagnosed ectopic tubal pregnancy. The cytologic method is easily performed and is rapid, sensitive, certain, inexpensive, and permanent. Cytologic study should not supplant meticulous macroscopic examination of abortion products by the clinician.

UDP-Glucuronate, formed through dehydrogenation of UDP-glucose and itself decarboxylated to make UDP-xylose, is the presumed donor for the glucuronyl side-groups in biosynthesis of the capsular polysaccharide in Cryptococcus neoformans. A specific radiochromatographic assay for UDP-glucose dehydrogenase shows that the enzyme is present in the cytosol. The enzyme is very labile but is stabilized by 25% glycerol. The enzyme is inhibited strongly by NADH (Ki,NADH = Km,NAD = 0.1 to 0.2 mM). It is also inhibited competitively by UDP-xylose (Ki = 0.3 mM). These results suggest that the rate of production of the capsular precursor, UDP-glucuronate, is controlled by the intracellular concentrations of an end product, UDP-xylose, and of a direct product, NADH. Capsule mutants have been screened for activity of this enzyme but none is clearly deficient.

Cell wall mannoprotein of nonpathogenic yeasts is surface exposed, since the cells are agglutinated by concanavalin A and antimannoprotein antibodies. However, nonencapsulated cells of Cryptococcus neoformans were agglutinated neither by concanavalin A nor by antimannoprotein antibodies. Immunogold electron microscopy located most mannoprotein in the inner cell wall. Chemical analysis of purified cell walls showed the lack of mannose, xylose, and galactose residues. These data indicate that cryptococcal mannoprotein recovered from the cultural supernatant is a nonstructural element of the cell wall.

A serine auxotroph of Neurospora requires exogenous serine to repress the arylsulfatase, suggesting that this enzyme is repressed by cysteine and not by methionine.

Six acapsular strains of Cryptococcus neoformans obtained by chemical mutagenesis failed to produce a capsule in vivo and were avirulent in mice following high dose intramuscular, intraperitoneal or intravenous inoculation. Peritoneal granulomas were observed in all animals inoculated with the acapsular mutants. These granulomas were characterized by a large central mass consisting of intact, degenerating and necrotic yeast cells. This was surrounded by concentric layers of a broad band of histiocytes, a narrow band of fibroblasts, and around the periphery, a mass of lymphocytes and plasma cells. These isolates did not revert to an encapsulated or virulent state after more than a year of subculturing or 18 passages through mice.