Acid phosphatases of Sporothrix schenckii

Sporothrix schenckii cells were grown on a medium containing yeast extract, neopeptone and glucose at 20 degrees C to obtain a mixture of mycelia and conidia, and at 35 degrees C to obtain yeast-like cells. The organism was maintained in the mycelial form, and its transformation to yeast at the higher temperature proceeded via conidia and 'intermediate cells' that then gave rise to yeast by a blastic mechanism. Cell-free extracts were analysed by PAGE at pH 8.0 and acid phosphatases (EC 3.1.3.2) were revealed by a sensitive detection reagent at pH 5.0. Mycelial, conidial and yeast extracts all had some acid phosphatase activity (M-I, C-I and Y-I) at the origin, although the proportion was highest for the yeast extracts. All of the bands that penetrated the gels had different electrophoretic mobilities. Mycelial and conidial extracts each had one other isoenzyme (M-II and C-II), while the yeast extracts had a total of five electrophoretically distinct acid phosphatases. Isoenzyme Y-II was further resolved into five closely related bands (Y-IIa to Y-IIe), the relative intensities of which varied with the phosphate nutrition of the yeast cells and the history of the extracts. The acid phosphatase isoenzymes were inhibited to various extents by sodium fluoride, L(+)-tartrate and phosphate, and showed interactions with citrate as opposed to acetate as the background buffer at pH 5.0.     

Cytochemical studies of the types and localization of acid phosphatases in the various regions of the midgut epithelium of Carausius morosus

Synopsis Cytochemical studies on the localization and substrate specificities of acid phosphatase activities in the epithelial cells of the midgut ofCarausius morosus have revealed the presence of two distinct types of phosphatases. Acid naphthol AS-BI phosphatase activity was present at particulate (lysosomal) sites in all regions of the midgut and its activity was particularly high in the pear-shaped organs. Acid -glycerophosphatase of low activity was present in the mid and posterior midgut regions, but was absent from the pearshaped organs. In the anterior region of the midgut, acid -glycerophosphatase activity could only be found associated with the concentrically laminated vesicles.     

Cytochemical localization of acid phosphatase in striated muscle

Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.     

Cytochemical localization of acid phosphatase in striated muscle

Summary Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.Supported in part by grants AI 17945 and HL 17747 from the United States Public Health Service, National Institutes of Health     

A new procedure for the ultrastructural localization of acid phosphatases

Summary The synthesis and properties of two new lead-containing diazonium chlorides are described. The use of these reagents for the electron microscopical localization of acid phosphatases in animal cells has been assessed.     

Identification of three chitin synthase genes in the dimorphic fungal pathogenSporothrix schenckii

Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA ofSporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1. program showed thatS. schenckii consistently clustered most closely withNeurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungusS. schenckii with the Pyrenomycetes of the Ascomycota.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.     

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.     

Cytochemical demonstration of phosphatases in membrane-recycling structures of endodermal cells

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.     

Cytochemical localization of a D-amino acid oxidizing enzyme in peroxisomes of Drosophila melanogaster

A peroxide generating oxidase is demonstrated cytochemically in the peroxisomes of adult and larval Drosophila melanogaster, Oregon R and Rosy-506 strains. This enzyme activity is demonstrable using D-pipecolate or D-proline, but not L-proline, as substrate and is inhibited by kojic acid. Thus this enzyme shares cytochemical characteristics with vertebrate D-amino acid oxidase.     

Expression of a Pho85 cyclin-dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii

Sporothrix schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environmental conditions such as cell density, temperature, and calcium. We identified a homolog of the Pho85 cyclin-dependent kinase (Cdk) that mediates cellular responses to environmental conditions in other organisms. By Western blot, three proteins containing the PSTAIRE motif, which characterize the cyclin-dependent protein kinases, were identified in S. schenckii. The gene encoding a Pho85 homolog, PhoSs, was identified and sequenced. The phoSs gene consists of 990bp, contains one intron, and encodes a protein of 306 amino acids. The S. schenckii Pho85 homolog shares features with Cdks, including the PSTAIRE motif, an ATP binding domain, and a serine-threonine kinase domain. By quantitative competitive RT-PCR, expression of the phoSs gene was found to decrease 30-fold during the yeast to mycelium transition. The addition of extracellular calcium accelerated the dimorphic transition and restored phoSs expression. These findings suggest PhoSs may participate in the control of the yeast to mycelium transition in S. schenckii.     

Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens

Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Here we developed and optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) system of S. schenckii for insertional mutagenesis. The transformation efficiency reached more than 600 transformants per 10⁶ conidia. Using this protocol enabled us to obtain a large number of T-DNA insertional mutants within a short experimental period. Several mutants with altered phenotypes were obtained during the transformation experiments. The mutants displayed mitotic stability. Transferred DNA (T-DNA) flanking sequences were cloned by thermal asymmetric interlaced PCR (TAIL-PCR). Our results demonstrated that the ATMT system can be an effective tool for insertional mutagenesis in S. schenckii. This is the first report of a suitable mutagenesis system which may provide valuable mutants and information for both forward and reverse genetics research in the future for this medically important fungus.