Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways.

Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.

Lysosomal dysfunctions associated with mutations at mouse pigment genes.

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.

The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

Progenitor cell defect correctable by bone marrow transplantation in five independent mouse models of platelet storage pool deficiency.

Two human platelet storage pool deficiencies (SPD), Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited and characterized by hypopigmentation, prolonged bleeding, and normal platelet numbers accompanied by a reduction of platelet dense granules. Seven independent and unique mouse pigment mutations regulated by separate genes have been proposed as animal models for SPD. Mice homozygous for the recessive mutations have diluted pigmentation, prolonged bleeding times, normal platelet concentrations, and reduced numbers of platelet dense granules. Reciprocal bone marrow transplantations were carried out between normal C57Bl/6J mice and five of these mutants, pearl, light ear, pale ear, ruby-eye, and maroon, to test whether the platelet defects are due to platelet progenitor cells or to humoral regulatory factors. Recipient mice were transplanted with marrow after 950-rad whole body irradiation. The prolonged bleeding time and low serotonin concentrations of the five mutants were converted to normal values after transplantation with normal marrow. Normal mice displayed characteristics of platelet SPD when transplanted with mutant marrow. This study demonstrates that in each of five independent mouse models the thrombopathy of SPD is due to a platelet progenitor cell defect correctable by bone marrow transplantation. These findings suggest that in severe cases human SPD may be amenable to treatment by bone marrow transplantation.

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Study of mold allergy in asthmatic children in Hungary.

We studied the change in sensitivity to propagating aerogenic fungi (spores, conidia) in extrinsic asthmatic children living in an urban environment from 1977 to 1988. According to the skin test, 10.6% of those examined in 1977 were sensitive to the fungi, the proportion being 30.4% in 1985 and 38.5% in 1987/88. The increase may be explained by the increasing frequency of sensitivity to Alternaria alternata and Phoma betae. In skin tests with Bencard allergens, reaction to both types was frequently observed. Of those sensitive to P. betae, 83% were also sensitive to A. alternata, and 87.5% of those sensitive to A. alternata were also sensitive to P. betae. The frequency of cross-reactions observed both with skin tests and specific IgE determinations suggests the presence of a common allergen, or epitope. The effect of environmental factors was analyzed with computer techniques. P. betae allergy was not related to detectable mold, humidity or number of pot plants in the home. The living conditions changed during the study period as follows: 1) housing conditions improved; 2) energy-saving building technologies were generally accepted; and 3) air pollution increased, also affecting the vegetation. The sensitizing masses of spores and conidia originated most likely from molds living on plants weakened and diseased by environmental pollution.

L-glutamine as a substrate for L-asparaginase from Serratia marcescens.

l-Asparaginase from Serratia marcescens was found to hydrolyze l-glutamine at 5% of the rate of l-asparagine hydrolysis. The ratio of the two activities did not change through several stages of purification, anionic and cationic polyacrylamide disk gel electrophoresis, and partial thermal inactivation. The two activities had parallel blood clearance rates in mice. l-glutamine was found to be a competitive inhibitor of l-asparagine hydrolysis. A separate l-glutaminase enzyme free of l-asparaginase activity was separated by diethylaminoethyl-cellulose chromatography.

Hygienic mycology in Hungary.

In Hungary the medical mycological research concerning the systemic (so-called deep) mycoses started in the National Institute of Hygiene. The Mycology Department has been founded in 1955 in this Institute with nation-wide authority as a routine diagnostic laboratory for clinical specimens from systemic mycotic infections. Three years later its activity was broadened--on ground of the experiences--to cover the total field of hygiene, thus we had to establish the "mycologia hygienica" (hygienic mycology), however, this has not been accepted as an independent discipline. Here a short, selected overview is given of our experiences. In the following the trends of the activity is demonstrated along the subdivisions of hygiene (epidemiology, environmental-hygiene, professional and occupational-, nutritional and food hygiene). In respect of hygiene, however, the fungi play a Janus-faced (more exactly Dr. Jekyll-Mr. Hyde) role, as they can behave not only harmful but also beneficial.

Correction of symptoms of platelet storage pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak syndrome.

Two human diseases of platelet storage pool deficiency (SPD), Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.

Inherited prolonged bleeding time and platelet storage pool deficiency in the subtle gray (sut) mouse.

A high proportion of mouse mutants with diluted pigmentation have severely prolonged bleeding times due to platelet storage pool deficiency. The deficiency is associated with concomitant abnormalities in platelet dense granules and coat pigment granules. The coat color of the subtle gray (sut) mouse is diluted to a relatively minor degree. Analysis of platelet serotonin concentration established that this dense granule component similarly is reduced a relatively small amount in this mutant. The subtle gray mouse thus allowed a test of the hypothesis that relatively small changes in platelet dense granule contents may cause discernible increases in bleeding times. Bleeding times of mutant mice were significantly prolonged (3.4-fold) in comparison with those in normal sut/+ controls. These bleeding times were significantly reduced in comparison with other mouse pigment dilution mutants with more severe storage pool deficiency. These results establish the subtle gray mouse as an appropriate animal model for mild storage pool deficiency and human Hermansky-Pudlak syndrome. They indicate, together with related experiments, that bleeding times are highly sensitive to concentrations of platelet dense granule components such as serotonin.

The macroconidial septum of some dermatophyton species.

The septum of the macroconidium (MC) of dermatophytes was examined by light microscopy. Significant taxonomical differences between species have not been found. The septa seem to be three-layered, but only the middle layer can be considered a septum in the wall substance of two neighbouring chambers, impressed and completed by the septum. The septum verum originates from the middle layer of the three-layered MC wall or from its surface adjacent to the chamber wall. This type of septum is different from that described in Ascomycetes and other species. Light microscopic findings are insufficient to characterize the nature of the septal pore. The microscopic observation of the layers of the MC wall and of the septum may be disturbed by misleading optical phenomena.

Some characteristics of nystatin-resistant sterol mutants of Candida albicans.

Various stable, auxotrophic and nystatin-resistant sterol mutants of Candida albicans were isolated after nitrosoguanidine treatment. Sterol mutants were divided into groups on the basis of the ultraviolet spectra and thin-layer chromatographic patterns of their nonsaponifiable sterol extracts. They were further characterized by their conductometrically measured nystatin-induced ion release. These sterol mutants displayed a decreased growth yield and an increased cell volume. On media containing 0.01% of the carbon sources, most of them could assimilate glycerol, alpha-methyl-D-glucoside, DL-lactic acid, L-sorbose, L-arabinose and ribitol only to a significantly reduced extent, or not at all. It is presumed that these properties result from the altered sterol composition of the plasma membrane.

Freeze fracture electron microscopical investigation of Candida albicans cells sensitive and resistant to nystatin.

A sterol mutant was isolated after nitrosoguanidine treatment, on the basis of th nystatin-resistant phenotypic behaviour, from a Candida albicans strain requiring adenine. The plasma membrane ultrastructure of the resistant mutant, observed by freeze-fracture electron microscopy, was not significantly different from the parental, ergosterol producing nystatin-sensitive strain. Nystatin treatment caused no ultrastructural change in the resistant strain, but led to the aggregation of membrane particles in the sensitive strain: this effect was considered to be the specific effect of nystatin. Other, probably nonspecific effects of nystatin on the sensitive strain were a deepening and deformation of invaginations, atypical membrane fracture, certain changes in the structure of cell wall and special ornamentation of its surface. No change in the intracellular membranes was observed.

The mouse pale ear pigment mutant as a possible animal model for human platelet storage pool deficiency.

The mouse pigment mutant pale ear, ep/ep, which has a defect in kidney lysosomal enzyme secretion, had prolonged bleeding on experimental injury. Platelet counts and platelet protein did not differ from normal. There was, however, a deficiency in the platelet dense granule contents, serotonin, ATP, and ADP. Furthermore, a marked reduction of platelet dense granules was observed by electron microscopy. The results suggest that pale ear is a useful animal model in the study of platelet storage pool disease. Studies on this mutant and other pigment mutants have established that one gene can regulate at least three subcellular organelles, including the melanosome, the lysosome, and the platelet dense granule.

Morphogenetic effect of L-cysteine on dermatophytes.

In the presence of L-cysteine, all the 24 dermatophyton fungi under study grew poorly. None of the strains, except Trichophyton menatographytes var. quinckeanum, grew in the presence of 0.04 M L-cysteine. The strains growing on a medium containing L-cysteine showed morphological changes. The surface of the colonies lost its velvety appearance and became awnless or waxy. The strains grown in the presence of L-cysteine abundantly formed chlamydospores. The chains of chlamydospores may resemble yeast cell chains, but true budding forms were not found in cultures in vitro. If strains precultivated on L-cysteine-containing medium were injected intraperitoneally into mice budding forms appeared in the peritoneal fluid.