Extracellular proteolytic enzyme activity ofHistoplasma capsulatum var.duboisii

Histoplasma capsulatum var.duboisii is the etiological agent of African histoplasmosis, an important deep mycosis in West Africa. Not much is known about the physiological properties of this fungus. This communication reports on the extracellular proteolytic enzyme activity of this fungus. Five isolates of this fungus tested hydrolyzed azocasein and bovine serum albumin at pH 6.8 and 8.0. Assay of the crude enzyme showed that proteolytic activity increased with age and peaked on the 10th day and then again on the 13th day for the yeast form, and on the 11th day of growth for the mycelial form. The optimum temperature and pH for maximum enzyme activity were 35 °C and 6.8 respectively. The proteinase activity was more pronounced with the yeast form than with the mycelial form. The action of enzyme inhibitors suggested the presence of an aspartyl proteinase.     

Two-dimensional immunoelectrophoresis of histoplasmin and a purified polysaccharide-protein antigen ofHistoplasma capsulatum

Crude histoplasmin and a polysaccharide-protein complex (PPC-histo) antigens obtained from culture filtrates ofHistoplasma capsulatum were analyzed by single and tandem two-dimensional immunoelectrophoresis (TD-IEP) using a rabbit hyperimmune anti-histoplasmin polyvalent serum. Single TD-IEP showed 14 arc precipitates for histoplasmin. Continuity of arcs 2, 6, and 7, and 9 and 10 was observed, suggesting a different polymeric configuration of the same antigen. This was also confirmed in tandem TD-IEP of histoplasmin with homologous (PPC-histo) and heterologous PPC's fromBlastomyces dermatitidis, Paracoccidioides brasiliensis andCoccidioides immitis. Tandem TD-IEP of histoplasmin and PPC-histo displayed a similar antigenic pattern to histoplasmin alone, being arcs 1 and 3 more evident and apparently present only in histoplasmin and PPC-histo. Tandem TD-IEP showed common antigens among the other heterologous fungal purified antigens, and seems useful to observe the multiplicity of antigens present in fungal preparations and to identify those precipitates (arcs 1 and 3) that are predominant in the purified preparation.     

Epididymal histoplasmosis diagnosed by isolation ofHistoplasma capsulatum from semen

An autochthonous case of epididymal histoplasmosis masquerading as tuberculosis in a 55-year-old male patient is reported from India. It was diagnosed by culture ofHistoplasma capsulatum from semen and by demonstration of the fungus upon re-examination of epididymal biopsy sections previously misinterpreted as tuberculous granuloma. The patient's main complaints were painful epididymal swelling, occasional fever and cough. He was treated successfully by excision of epididymis and vas deferens combined with amphotericin B therapy. This is believed to be the first case of epididymal histoplasmosis to be reported outside the American continent and the fourth of its type reported in the English literature. The case is also noteworthy in thatH. capsulatum was isolated for the first time from semen, and it underlines the importance of mycological culture of semen specimens for diagnosis of genitourinary infections of obscure etiology.Presented at the XII Congress of the International Society for Human and Animal Mycology, Adelaide, Australia, March 13–18, 1994.     

The asteroid body of lobomycosis

The epidemiology of histoplasmosis duboisii (African histoplasmosis) is not well understood. The present study was carried out to investigate the prevalence of skin sensitivity and to determine by immunodiffusion the presence of antibodies among humans to histoplasmin around a recently discovered natural focus of Histoplasma capsulatum var. duboisii in a bat cave in Ogbunike in the Anambra State of Nigeria. Out of the 40 subjects, all young adults aged 18–30 years, comprising cave guides, traders and farmers examined in the immediate vicinity of the cave, 14 (35.0%) gave a positive skin test. In another population of the same age group, comprising 620 persons, viz. traders, farmers, palm oil workers and some patients attending rural clinics, examined in other nearby areas in Anambra State, 55 (8.8%) reacted positively to histoplasmin. In the immunodiffusion tests, 2 (2.08%) of the 96 school children and 17 (9.4%) of the 181 young adults, including farmers, palm oil workers and traders tested amongst the population around the cave, demonstrated precipitating antibodies to histoplasmin in their sera. Only 5 (0.79%) of the 630 adults of the same age group with similar occupations examined from other areas in Anambra State had precipitating antibodies. Out of another 50 subjects examined, viz.; wood workers, traders, farmers, and school teachers in Nsukka in the Enugu State, two (4.0%) demonstrated antibodies. It is suggested that asymptomatic infections due to the duboisii variety of H. capsulatum may be common in the human population around the cave. A diligent search with the help of local hospitals and public health officials may reveal clinical cases of histoplasmosis duboisii with cutaneous and systemic lesions.     

Histoplasma capsulatum peritonitis associated with continuous ambulatory peritoneal dialysis

This paper reports a case of infection byHistoplasma capsulatum apparently restricted to the peritoneum in a woman submitted to continuous ambulatory peritoneal dialysis. Diagnosis was established by culture of dialysis fluid and peritoneal nodule and by histopathologic examination.     

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy

Summary We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/g DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/gm DNA).     

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall ofHistoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.Abbreviations CHHA Cystine-heart-hemoglobin agar - PBS phosphate buffered saline - Ig immunoglobulin - TBS Tris buffered saline - DAB 3,3-diaminobenzidine tetrachloride - FITC fluorescein isothiocyanate - M199 tissue culture medium 199, according to Morgan et al. (1950)     

Paraquat induced thiol modulation of Histoplasma capsulatum morphogenesis

Cysteine metabolism with the subsequent release of anionic thiols has been shown to be involved in yeast cell morphogenesis of the dimorphic, pathogenic fungus Histoplasma capsulatum. Following transfer to fresh medium, intracellular thiol levels during the initial 2–4 h appear to determine the eventual growth form. Mild oxidative stress induced by paraquat (methyl viologen) caused enhanced intracellular and extracellular thiol production and an increase in protein thiol formation. Mildly stressed cells continued to grow in the yeast form. Severe oxidative stress induced by high concentrations of paraquat resulted in lowered thiol production as well as reversion to the alternate mycelial morphology. These results suggest that thiol modulation of intracellular protein status may be involved in morphogenesis of H. capsulatum.     

Immunosuppression transfer by spleen cells from young to adult mice previous to Histoplasma capsulatum infection

The passive transfer of spleen cells from 1 month old mice into adult syngeneic mice, abrogates their resistance to histoplasmal infection. This suppressive state was detected in two cell populations, one non-adherent and another adherent with radioresistant characteristics.The transferred spleen cells were treated by different anti-sera: anti-theta, anti-adherent cells (produced in rabbits) and monoclonal anti-Thy 1.2 respectively.The irradiated and non-irradiated adult recipient mice were infected with Histoplasma yeasts utilizing the Lethal Dose₅₀ for 1 month old mice. The infection course was determined by death percentage, the histoplasmosis murine signs and the number of the fungal colony forming units (CFU) from the infected spleens. The results of the anti-sera treatment suggest that non-adherent as well as adherent cells participate in the suppressive phenomena. A lower number of CFU was identified in infected animals which received cells treated with anti-Thy 1.2 anti-sera.     

Staurastrum dilatatum var.thomassonii var. nov.

The new varietyStaurastrum dilatatum Ehr. var.thomassonii is described and illustrated.     

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Summary Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.     

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU colony forming units - PMN polymorphonuclear neutrophil - CTCM complete tissue culture medium - CL chemiluminescence - HPO horseradish peroxidase - P-L lysosomal peroxidase positive material     

Induction of novel protein synthesis by opsonizedHistoplasma capsulatum ingested by murine peritoneal macrophages

It is known thatHistoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closly related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form ofH. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingestedH. capsulatum with [³⁵S]-methionine labeling. Cycloheximide at 5 to 10 µg/ml was used to preferentially inhibit macrophage uptake of [³⁵S]-methionine without affectingH. capsulatum uptake. Protein synthesis byH. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killedH. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingestedH. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingestedH. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.     

First environmental isolation of Cryptococcus neoformans var. neoformans and var. gatti from the Gharbia Governorate,Egypt

Flowers from two Eucalyptus camaldulensis trees in the Qutur area and one tree from the Tanta area yielded three isolates of Cryptococcus neoformans var. gattii. Pigeon and sparrow droppings were also investigated for the occurrence of C. neoformans within the study area. Ninety five isolates of the neoformans variety of C. neoformans were recovered from 550 samples of avian droppings. This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparative study of leaf carbon gain in saplings ofThujopsis dolabrata var.hondai andQuercus mongolica var.grosseserrata in a cool-temperate deciduous forest

Seasonal courses of leaf CO₂ gas exchange in a growing season were examined in saplings ofThujopsis dolabrata var.hondai andQuercus mongolica var.grosseserrata in a cool temperate deciduous forest. Between the two tree species there were no large differences in the light compensation point of leaf photosynthesis, except for the season of new leaf expansion. However, light-saturated rates of net photosynthesis were obviously high inT. dolabrata var.hondai. EvergreenT. dolabrata var.hondai saplings had large photosynthetic production in two seasons, before the emergence of new foliage and after foliage fall of the overstory deciduous trees, because of the significantly high solar radiant energy penetrating under the forest canopy during the seasons. Saplings of deciduousQ. mongolica var.grosseserrata were heavily shaded throughout the growing season by foliage of the overstory trees, which resulted in a low daily surplus production. The annual surplus production of leaves in the growing season was estimated to be 2300 mmol CO₂ m⁻² inT. dolabrata var.hondai and −100 mmol CO₂ m⁻², slightly negative, inQ. mongolica var.grosseserrata. These results supported the high survivability ofT. dolabrata var.hondai saplings and the high mortality ofQ. mongolica var.grosseserrata in the deciduous forest.     

Sporobolomyces salmonicolor var. fischerii,a new yeast

A new variety of Sporobolomyces salmonicolor, a basidiomycetous yeast isolated from the cerebrospinal fluid of a human case of meningitis has been described. The variety can be differentiated from the species primarily by its ability to assimilate D-galactose and L-arabinose.     

A new genus of Platycopioida (Copepoda) from a marine cave on Bermuda

Nanocopia minuta was collected from an inland marine cave on Bermuda. This new genus is reminiscent of Platycopia in its cephalic appendages and in the 5th legs of the male. The 1st leg has a 3-segmented exopod and a 1-segmented endopod. The other legs show reductions with no armature along the inner margin of the exopods and with 2-segmented endopods in the female. Only 2nd and 3rd legs bear two outer spines on the first exopodal segment.Owing to similarities in sexual characters, mouthparts, and modifications of the swimming legs, Nanocopia and Platycopia are considered more closely related to each other than either is to Antrisocopia.Platycopioida now contains three genera of which two are found only in Roadside Cave. The order has retained several primitive characters and seems to have separated early from the gymnoplean stem.     

珍稀濒危植物长白松(Pinus sylvestris var. sylvestriformis)天然种群生存压力

为了揭示珍稀濒危植物长白松（Pinus sylvestris var. sylvestriformis）天然种群生存压力状况,在全面调查长白山国家级自然保护区长白松分布的基础上,基于邻体干扰模型,引入树高、冠幅、方位等因子,提出3种生存压力指数：个体生存压力指数、种群生存压力指数和群落生存压力指数,分析天然长白松所处6种群落类型中的生存压力。结果表明：长白松承受群落生存压力（PI）从大到小依次为：白桦-臭冷杉群落（PI=21.532）、红松-长白松群落（PI=14.185）、白桦群落（PI=13.262）、臭冷杉-长白松群落（PI=8.752）、长白落叶松-鱼鳞云杉群落（PI=7.780）和蒙古栎群落（PI=5.440）。多重比较单向方差分析表明,6种群落类型中长白松生存压力总体上差异明显,白桦-臭冷杉群落中长白松生存压力最大,显著高于其他5种群落;竞争树种主要为长白落叶松、红松、长白松、山杨和白桦,这5个树种生存压力大小占群落生存压力的87%;红松-长白松群落和白桦群落中长白松生存压力无明显差异,但显著高于臭冷杉-长白松群落、长白落叶松-鱼鳞云杉群落和蒙古栎群落;臭冷杉-长白松群落、长白落叶松-鱼鳞云杉群落和蒙古栎群落中长白松生存压力相对较小,彼此无明显差异。长白松生存压力与其所处植物群落演替阶段及其龄级结构有关。目前,保护区采取严格保护和管理方式不完全有利于长白松种群的稳定发展。根据长白松种群所处的植物群落生境特点、种群生存压力状况并结合种群年龄结构特征,针对不同群落类型提出相应抚育措施建议以期为长白松天然种群的保护提供参考。