Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca²⁺. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.     

Insect haemolymph: Cooperation between humoral and cellular factors in Locusta migratoria

In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 min. Both lipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 min prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.     

Using volatile organic compounds to enhance atrazine biodegradation in a biobed system

The effect of the terpenes α-pinene, eucalyptol, and limonene, individually and as mixtures, on atrazine (ATZ) biodegradation and on biological activity in a biobed biomixture was evaluated. Additionally, terpenes emitted from the biomixture were captured using solid-phase microextraction. Terpenes added individually at relatively low concentrations (50 μg kg^?1) significantly enhanced ATZ degradation and biological activity during the first incubation days. No significant effect on ATZ degradation was found from adding the terpene mixture, and, interestingly, an inhibitory effect on phenoloxidase activity was found during the first 20 days of incubation when mixed terpenes were present at 100 μg kg^?1. Capturing terpenes demonstrated that during the first hour of incubation a significant fraction of the terpenes was volatilized. These results are the first to demonstrate the feasibility of using terpenes to enhance the degradation of a pesticide. However, successive applications of terpenes or the addition of materials that slowly release terpenes could sustain the ATZ degradation enhancement.     

Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

Interactions between the endocrine and immune systems in locusts

Abstract. The prophenoloxidase cascade in the haemolymph of mature adult Locusta migratoria migratorioides (R & F) is activated in response to injection of laminarin, a β‐1,3 glucan. Co‐injection of adipokinetic hormone‐I (Lom‐AKH‐I) and laminarin prolongs the activation of the enzyme in a dose‐dependent manner. However, injections of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase unless AKH is co‐injected, when there is a dose‐dependent increase in the level of phenoloxidase that persists in the haemolymph for several hours. Even when AKH is co‐injected, the highest levels of phenoloxidase activity are always greater after injection of laminarin than after LPS, and these two immunogens must activate the prophenoloxidase cascade by quite distinct pathways. In the present study, interactions between the endocrine and immune systems were examined with respect to activation of prophenoloxidase and the formation of nodules: injection of LPS induces nodule formation in adult locusts. With LPS from Pseudomonas aeruginosa, nodules form exclusively in dense accumulations in the anterior portion of the abdomen on either side of the dorsal blood vessel associated with the dorsal diaphragm. However, with LPS from Escherichia coli, fewer nodules are formed but with a similar distribution, except that occasionally some nodules are aligned additionally on either side of the ventral nerve cord. Co‐injection of Lom‐AKH‐I with LPS from either bacteria stimulates greater numbers of nodules to be formed. This effect of coinjection of AKH on nodule formation is seen at low doses of hormone with only 0.3 or 0.4 pmol of Lom‐AKH‐1, respectively, increasing the number of nodules by 50%. Injections of octopamine or 5‐hydroxytryptamine do not mimic either of the actions of Lom‐AKH‐I described here. Co‐injection of an angiotensin‐converting enzyme inhibitor, captopril, reduces nodule formation in response to injections of LPS but has no effect on the activation of phenoloxidase. Co‐injection of an inhibitor of eicosanoid synthesis, dexamethasone, with LPS influences nodule formation (with or without AKH) in different ways according to the dose of dexamethasone used, but does not affect activation of prophenoloxidase. Eicosanoid synthesis is important for nodule formation, but not for the activation of the prophenoloxidase cascade in locust haemolymph.     

Phagocytic release of human leukocyte neutral proteases: Analysis of cartilage degradation products

Human polymorphonuclear leukocyte neutral proteases (HLNP) released during the process of phagocytosis of aggregated human gamma globulin were tested for their ability to degrade intact rabbit ear cartilage. Using ³⁵S-labeled cartilage as substrate, HLNP derived from 45 × 10⁷ cells released about 45% of the total incorporated ³⁵S. DE-52 chromatography of incubation supernatants revealed a single ³⁵S peak associated with minimal quantities of peptide or protein material as estimated by absorbance at OD_{230 + 280 nm}. Analytical ultracentrifugation gave a molecular weight of 51,800. Incubation of cartilage with excess α-chymotrypsin released ³⁵S-containing protein and peptide elements (M_r 79,400). Therefore, degradation, of the proteoglycans of intact cartilage by HLNP is more extensive than that noted with bovine pancreas α-chymotrypsin. The products of HLNP and α-chymotrypsin digestion of cartilage contained chondroitin sulfates A and/or C since both materials (after column chromatography) were sensitive to chrondroitinase ABC and AC digestion. Collagenolytic activity of HLNP is minimal compared to proteolytic activity as evidenced by the lack of hydroxyproline containing peptides released from cartilage during enzyme incubation. It is suggested that HNLP incubated with intact cartilage may serve as a relevant model in the search for new agents which could combat enzyme-mediated cartilage destruction.     

Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Suppressive effects of metabolites from Photorhabdus and Xenorhabdus spp. on phytopathogens of peach and pecan

Abstract

Our objective was to determine the suppressive abilities of bacterial metabolites derived from Xenorhabdus and Photorhabdus spp. on Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, and Fusicladosporium effusum, which are fungal or oomycete pathogens of pecan, and Monilinia fructicola, a fungal pathogen of peach. In the first set of in vitro assays, when metabolites were compared based on initial bacterial cell count, X. bovienii (SN) metabolites generally exhibited the greatest suppression of phytopathogens and Xenorhabdus sp. (355) the least with Photorhabdus luminescens (Hb) and Xenorhabdus nematophila (All) being intermediate. In a second set of in vitro assays, in which metabolites were compared at 50 mg per ml acetone, P. luminescens (VS) exhibited greater suppression than P. luminescens (Hb), Photorhabdus sp. (MX4), X. bovienii (SN), and Xenorhabdus sp. (3 – 8b). In in vivo tests, 6 or 12% dilutions of X. bovienii (SN) or P. luminescens (Hb) metabolites caused 90 – 100% suppression of P. cactorum lesions on pecan leaves with only slight phytotoxicity. No phytotoxic effects were observed in detached peach leaves at dilutions up to 25%. Metabolite treatments, derived from X. bovienii (SN) and P. luminescens (Hb), were also tested for suppression of F. effusum sporulation in detached pecan shoots. Reductions in sporulation caused by bacterial metabolites were similar to those following treatment with two chemical fungicides, dodine and fenbuconazole; a third chemical triphenyltin hydroxide had no effect. Further research is warranted to determine if fungal or oomycete incited diseases in pecan and peach can be controlled with metabolites of Xenorhabdus spp. and Photorhabdus spp.     

Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.     

Involvement of larvicidal toxins in pathogenesis of insect parasitism with the rhabditoid nematodes,Steinernema feltiae andHeterorhabditis bacteriophora

The insect-parasitic rhabditoid nematodes,Steinernema feltiae andHeterorhabditis bacteriophora, released a compound/s/ toxic to larvae of the greater wax moth,Galleria mellonella, that caused paralysis and death of the insect. Larvicidal substances appeared in wax moth larvae during parasitism and after inoculation with the primary form of the bacterial associates of the nematodes. The nematodeS. feltiae and its associate,Xenorhabdus nematophilus, excreted much less toxic activity within larval body thanH. bacteriophora. The secondary form ofXenohabdus did not produce toxin in parasitized larvae, butX. luminescens, the bacterium associated withH. bacteriophora, released detectable titer of toxin activity in broth cultures. Both nematode toxins were sensitive to heat and produced a specific type of proteolytic activity. Preliminary identification of the compounds responsible for larval toxicity revealed similarities to immune inhibitors produced by some bacterial pathogens of insects.      

Temperature and Ca2+ ion as modulators in cellular immunity of the Sunn pest Eurygaster integriceps Puton (Heteroptera: Scutelleridae)

Environmental conditions in addition to divalent cations may affect the interactions between pathogens and insects. Elucidation of factors which modulate insect immune responses could be a significant part of investigations in this area. In this study, adults of Eurygaster integriceps, as the destructive pest of wheat, were kept at different temperatures in addition to injection with different concentrations of Ca²⁺ to find the effect on cellular immune reactions against Beauveria bassiana. Results showed that total and differentiate hemocyte numbers, nodule formation and phenoloxidase activity increased with elevation of temperature so that the higher values were obtained at 30 and 40°C at various intervals. Higher concentrations of Ca²⁺ ion (5 mM) caused an increase in plasmatocyte length and width especially after 60 min. Similar results were observed for nodule formation and phenoloxidase activity of E. integriceps adults after injection by B. bassiana spores and phenoloxidase activity. It is clear from the current study that thermoregulation and Ca²⁺ ion can positively affect the hemocyte numbers especially plasmatocytes and granulocytes, nodule formation and phenoloxidase activity in E. integriceps. The understanding of modulators of the insect immune response may directly influence novel approaches to obtain safe and effective biological control agents.     

Changes of the Antioxidant Status and System of Generation of Free Radicals in Hemolymph of Galleria mellonella Larvae at Microsporidiosis

Changes of superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities as well as of the content of SH-containing compounds were revealed in hemolymph of the native and the Vairimorpha ephestiae microsporidian-infected greater wax moth Galleria mellonella larvae. The SOD and GST activities in hemolymph of infected insects decreased at the stage of merogony, whereas during massive sporulation the enzymatic antioxidant activity in host tissues was higher than in control. By the ESR spectroscopy method, using the 1-hydroxy-3-carboxy-pyrrolidinespin-trap, generation of free radicals in hemolymph of infected insects was shown to decrease only at the stage of sporogony. The phenoloxidase activity in lymph was lower at acute microsporidiosis than in native larvae. The hemolymph concentration of thiol-containing proteins in infected insects did not differ from that in control. We suggest that decrease of generation of free radicals in hemolymph of the greater wax moth larvae at the stage of sporogony is due to a suppression of the prophenoloxidase system and an elevation of the antioxidant activity.     

A Bowman–Birk protease inhibitor with antifeedant and antifungal activity from Dolichos biflorus

In the present study, 11 varieties of Dolichos biflorus exhibited both protease inhibitor activities as well as in vitro inhibitory activity against Helicoverpa armigera gut protease. A Bowman–Birk protease inhibitor showing activity against trypsin and α-chymotrypsin has been purified from D. biflorus seeds using multi-step strategy. The purified inhibitor revealed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa. The inhibitory constants for the interaction of purified PI with trypsin and α-chymotrypsin were 0.04 and 0.48 μM, respectively. The purified inhibitor was stable over a pH range of 2–12 and up to a temperature of 100 °C for 20 min. The results of insect bioassay against H. armigera revealed 68 % decline in larval weight after 7 days of feeding on artificial diet containing the inhibitor. The larval growth and % leaf area eaten were drastically reduced in the presence of inhibitor. The observed cumulative mortality from larval to adult was 51.21 %. The inhibitor displayed antifungal activity against Alternaria alternata, Fusarium oxysporum, and Aspergillus niger with minimum inhibitory concentration as 0.4, 0.6, and 1.2 μg mL^?1, respectively. This is the first report of anti-feedant and anti-fungal activities of D. biflorus protease inhibitor on a single protein, which might be important for developing transgenic plants resistant to insect pests and fungal pathogens.     

Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus

Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of X. nematophilus subsp. dutki (isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of G. mellonella larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with X. nematophilus was stable at 60°C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27°C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of X. nematophilus. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of X. nematophilus and the time of sampling.     

Neoaplectana species: Specificity of association with bacteria of the genus Xenorhabdus

Each of five Neoaplectana (Nematoda: Steinernematidae) species was cultured monoxenically with various Xenorhabdus (Eubacteriales: Enterobacteriaceae) isolates. The nematodes were usually able to reproduce when cultured with the bacterial symbiont of any one of the five Neoaplectana spp. but never with Xenorhabdus luminescens, symbiotic with Heterorhabditis spp., or with the Xenorhabdus sp. isolated from an undescribed steinernematid species. Only Neoaplectana bibionis could be cultured with the Xenorhabdus symbiont of Steinernema kraussei. A high proportion of infectives were able to carry within their intestine X. nematophilus isolated from other strains of the same nematode species; a small proportion of infectives were able to carry X. nematophilus isolated from other nematode species.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

Effect of different K+ concentrations on cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe³⁺ and Cu²⁺ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron [1–3]. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K⁺ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomlychosen, were tested for phenoloxidase activity, according to the modified protocols of Polachecket al., Torres-Guerrero et al. and Rhodes [2–4]. According to the results obtained, it has been observed that K⁺ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.This revised version was published online in October 2005 with corrections to the Cover Date.