A mutation (dosach) in Drosophila which affects aster formation and nuclear migration during cleavage.

Here we describe a new mutant, dosach (dos), in Drosophila melanogaster. In the mutant, centrosomes divide and initiate spindle formation similar to that seen in wild-type embryos. Nevertheless, mutant embryos form cleavage spindles that lack visible asters and display abnormal morphology, including mono- and tri-polar spindles, spindle chains and incorrect alignment. Irregular nuclear migration is also observed in mutant embryos, and this may suggest that astral microtubules are important for spindle spacing during cleavage and also in maintaining the integrity of the mitotic apparatus. Confocal microscopy has been used to correlate organization of microtubules, centrosomal proteins and chromosomes in wild-type and dosach (dos) embryos.

Identification and partial characterization of a unique marker for human basophils.

A unique marker for human basophils is needed to precisely determine the involvement of this cell type in clinical disease. To search for a marker of the basophil secretory granule, mouse hybridomas were generated against purified human basophils and screened for basophil-selective Ab. One hybridoma (2D7) produced an IgG1 kappa Ab that labeled basophils, but not lymphocytes, monocytes, eosinophils, neutrophils, and mast cells by an indirect immunoperoxidase procedure. The pattern of basophil staining was cytoplasmic and granular by light microscopy. By immunogold electron microscopy, the 2D7 ligand was localized to secretory granules. Activated basophils showed reduced 2D7-dependent staining intensity, consistent with a secretory granule localization. Tissue sections of normal skin, lung, and bowel showed no reactivity with 2D7, consistent with the anticipated absence of basophils in these tissues. 2D7 staining of basophils was clearly distinct from metachromatic staining, which was presumably dependent on proteoglycan. Extracts of normal human basophils subjected to Western blotting with 2D7 exhibited two predominant bands at apparent molecular masses of 76,150 and 72,260 Da. In summary, the 2D7 ligand appears to be a specific marker for human basophils and may facilitate the assessment of basophil involvement in diseases such as asthma, anaphylaxis, and atopic dermatitis.

Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils.

Galectin-3 is an endogenous soluble lectin within the family called galectins that bind beta-galactosides. Homologs of the protein isolated from different sources were previously designated as IgE-binding protein (epsilon BP), CBP35, CPB30, Mac-2, RL-29, RLL, L-29, and HL-29. All are now renamed galectin-3. This lectin is widely distributed in cells and tissues of mice, rats, dogs, hamsters, and humans. Light microscopic immunohistochemistry and ultrastructural immunogold labeling methods were used to determine the distribution of galectin-3 in human mast cells of several organs, in mast cells developed in vitro from human fetal liver cells, and in human peripheral blood basophils. Immunolabeling for the protein was observed in mast cells from all sources and in basophils. The lectin was detected in the nucleus and/or the cytoplasm. The nuclear labeling was over heterochromatin whereas euchromatin was unlabeled. Cytoplasmic labeling was concentrated over secretory granules. The intensity of staining generally was greater in mast cells of skin when compared with that of mast cells in other locations and with that of basophils. Studies have indicated that in mast cells galectin-3 may be involved in promoting their adhesion to basal laminae. In this study the localization of galectin-3 in the secretory granules of human mast cells and basophils suggests that these cells may release this lectin when activated to degranulate.

Ultrastructural localization of heparin to human mast cells of the MCTC and MCT types by labeling with antithrombin III-gold.

BACKGROUND: Mast cells derived from human skin and lung have been reported to produce heparin and chondroitin sulfate E proteoglycans. However, no information about the proteoglycans distribution among the different human mast cell types (MCTC and MCT) is available. Conjugates of antithrombin III-gold were used to assess the presence of heparin in both human mast cell subsets. EXPERIMENTAL DESIGN: Thin sections of human and rodent tissues and dispersed cell preparations were labeled with the conjugate in the presence of saline, heparin, and chondroitin sulfates A and E and particle densities were measured over granules, perigranular regions, and extracellular space. Control sections were preincubated with heparinase, chondroitinase ABC, or buffer. RESULTS: Labeling with antithrombin III-gold particles was detected in essentially all granules of human mast cells in skin (predominantly MCTC type), lung alveolar wall, and bowel mucosa (predominantly MCT type), but was negligible over human eosinophils. Consistent with the known distribution of heparin in rodent mast cells, strong labeling was observed over rat peritoneal connective tissue type mast cells, but not over mucosal mast cells in bowel mucosa of Nippostrongylus brasiliensis-infected rats (which contain chondroitin sulfate di-B) nor over mouse PT-18 mast cells (which contain chondroitin sulfate E). Mast cell labeling was preferentially blocked by exogenous heparin, and virtually abolished by heparinase but not chondroitinase ABC preincubation. CONCLUSIONS: The data with rodent mast cells indicate that antithrombin III-gold labels cells that contain heparin, but not those that contain only over-sulfated chondroitin sulfates. Specificity of the procedure for detecting heparin is further demonstrated by inhibition of labeling after preincubation with heparinase and by competition with exogenous heparin. On this basis, we conclude that heparin is present in essentially all mast cells in normal skin, lung alveolar wall, and bowel mucosa. The presence of heparin in all human mast cells is different than for rodent mast cells, and probably accounts for the inability to clearly distinguish different human mast cell types from one another with histochemical stains based on proteoglycan content.

The adult who is an only child: achieving separation or individuation.

60 women and 60 men between the ages of 18 and 45 years (M = 30.5, SD = 9.6) were categorized by sex, age, and birth order (only child, firstborn, lastborn) to assess the differences among the adult only-child, the youngest child, and the oldest child in autonomous characteristics and cohesiveness in family interaction. Analysis of the responses on a biographical data sheet, the California Psychological Inventory, and the Family Adaptability Cohesion Scales III showed that main effects for birth order and sex are significant in the process of separation-individuation and that the only child is less autonomous than the oldest child.

Localization of rat tryptase to a subset of the connective tissue type of mast cell.

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.

The evaluation of hospice home care volunteers.

A national sample of 139 hospice volunteer coordinators rated a series of statements about volunteer behaviors in terms of their importance to volunteer performance. Each statement had to be rated at least 8 on a scale of 10 by 75% of the respondents in order to be included in the final instrument. This procedure yielded a scale of 27 descriptors of appropriate volunteer behavior. The scale is intended for use by hospice volunteer coordinators who want a tool with which to offer concrete, behavior based, constructive feedback to volunteers about their performance.

Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells.

We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation.

Sex-related differences in meprin-A, a membrane-bound mouse kidney proteinase.

To investigate the expression of meprin-A, a brush-border metalloproteinase in mouse tissues, immunohistochemical studies were conducted using a monoclonal antibody prepared against a purified form of kidney meprin-A form male mice. Kidney slices from female mice displayed markedly less immunoreactivity compared with similar preparations from male mice using this antibody. However, the specific activities of meprin-A in kidney homogenates and purified preparations of meprin-A from male and female mice were not significantly different. Western blots of kidney membrane proteins from several mouse strains indicated that the female form of meprin-A had a decreased mobility relative to the male form when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this difference could be eliminated by treatment of preparations with endoglycosidase F, which removes some asparagine-linked oligosaccharides. These data and lectin blots of membrane proteins indicate that there are differences in the glycosylation (specifically in the complex type oligosaccharides) of meprin-A in adult (8 wk old) male and female mice. Juvenile (3 wk old) male and female mice displayed similar amounts of immunohistochemical staining in kidney slices, as well as similar meprin-A electrophoretic mobilities and lectin affinities. Administration of 17 beta-estradiol to gonadectomized adult mice decreased the immunoreactivity of meprin-A in kidney slices and the electrophoretic mobility of meprin-A. These studies indicate that estrogens affect posttranslational modifications of meprin-A.

Immunohistochemical localization of the metalloproteinase meprin in salivary glands of male and female mice.

Meprin is a membrane-bound metalloproteinase which is expressed at high levels in the brush border membrane of proximal tubules of kidneys of some mouse strains (referred to as high meprin-activity mice). The mature active proteinase is not present in kidneys of many inbred strains of mice; however, these low meprin-activity mice possess a kidney protein that crossreacts with polyclonal antibodies prepared against meprin. In the present studies, immunohistochemical methods were used to determine the presence of meprin in liver, pancreas, spleen, testis, thymus, kidney, salivary glands, stomach, duodenum, and skin. Meprin crossreactivity was observed only in kidney and salivary glands. In salivary glands, the enzyme was found on the luminal surface of intercalated and striated ducts of submandibular and parotid glands and on interlobular ducts of the latter. In both kidney and salivary glands, the intensity of immunochemical staining was greater in males compared with females. For both sexes, immunoreactivity was markedly greater in the high meprin-activity mice compared to the low meprin-activity mice. These studies indicate that meprin has a limited tissue distribution, and that genetic and hormonal factors that regulate the proteinase are similar in kidney and salivary glands. The localization of the proteinase implies that the enzyme functions in modifying proteins and peptides that are secreted or re-absorbed in the ducts of these tissues.

Human MCTC type of mast cell granule: the uncommon occurrence of discrete scrolls associated with focal absence of chymase.

Two types of mast cells were previously defined based on neutral protease composition and ultrastructurally distinguished by granule morphology. The MCT cell contains tryptase with little, if any, chymase and was noted to have varying numbers of irregularly-shaped granules with discrete scrolls or particulate or beaded material. The MCTC cell contains both tryptase and chymase and was noted to have more regularly-shaped electron-dense granules with characteristic grating or lattice substructures. This study reports the use of electron microscopy and immunogold staining with antibodies against tryptase and chymase to demonstrate in mature unstimulated MCTC cells in situ, the focal occurrence of discrete or complete scrolls in peripheral regions of certain granules where chymase is deficient. these scrolls often appeared to be protruding from the granule. Granules containing discrete scrolls were observed in 10 of 340 mature MCTC cells, accounting for less than 1% of MCTC granules. Other granules in such cells as well as other regions of the granule under consideration, showed strong staining for both tryptase and chymase. These results strengthen the association of morphology with protease composition in human mast cell secretory granules, but weaken the use of morphology alone to identify the MCTC and MCT types of human mast cells. Whether the uncommon occurrence of focal absence of chymase in MCTC cells arises by chance or as a result of factors relating to mast cell development, interconversion, activation, or regranulation will require further clarification. In conclusion, the appearance of grating or lattice structures in mast cells indicates the presence of chymase and tryptase, characteristic of the MCTC phenotype, whereas multiple discrete scrolls in irregularly shaped granules suggests the MCT phenotype.

Ultrastructural analysis of maturing human T and TC mast cells in situ.

Mast cells at immature stages of development were identified in human tissues by electron microscopic techniques. General morphologic criteria of immaturity (such as a high apparent nuclear:cytoplasmic ratio and small cell size), the presence of few granules (those present being smaller than those in mature mast cells) and a lack of features of mast cell activation were used together to determine the level of maturity. Mast cells were identified as being of the T or TC type by immunogold staining with polyclonal rabbit IgG anti-chymase and murine monoclonal anti-tryptase primary antibodies and the appropriate gold-labeled secondary antibodies. Only those cells with tryptase-positive granules were recognized as mast cells. Immature T mast cell granules contained the same characteristic discrete scrolls found in their mature counterparts and all stained positive for tryptase. The presence of trace amounts of chymase in a minority of these granules, as in mature T mast cells, could not be ruled out. The majority of granules in immature TC mast cells had one or more amorphous electron-dense cores rather than the grating and lattice substructures characteristic of granules in mature TC mast cells. Secretory granules in immature TC mast cells stained positively for tryptase and chymase. Occasional immature TC mast cells contained a complete granule or a portion of a granule with the substructure characteristic of mature TC mast cells, favoring the concept that these TC mast cell forms are developmentally related. Essentially all mast cells in foreskin of newborns appeared immature, whereas 10, 5, 10, and 15% of the mast cells in adult lung, foreskin, bowel mucosa and bowel submucosa, respectively, appeared immature. The distribution of T and TC types of immature mast cells seemed to parallel that of the mature mast cell types. These compositional and ultrastructural differences between immature T and TC types of mast cells suggest that from the time granule formation begins, and possibly before this time, each type of human mast cell follows a distinct developmental pathway.

Ultrastructural analysis of human T and TC mast cells identified by immunoelectron microscopy.

Tryptase and chymase were localized in human mast cells by immunoelectron microscopy, enabling the T (tryptase positive, chymase negative) and TC (tryptase positive, chymase positive) types of mast cells to be identified and ultrastructurally characterized. A double immunogold staining procedure was performed on samples of human skin, small intestine, and lung with rabbit polyclonal IgG anti-chymase and mouse monoclonal IgG anti-tryptase primary antibodies and gold-conjugated secondary antibodies. Approximately 225 mast cells were examined in this fashion; comparable sections from 170 of these mast cells along with approximately 200 additional mast cells also were examined using techniques optimized for ultrastructural detail. Each secretory granule of TC mast cells contained both tryptase and chymase; secretory granules of T mast cells stained strongly positive for tryptase alone. Extremely small amounts of chymase appeared to be present in an occasional T mast cell granule. Staining for the neutral proteases was more intense over electron-dense regions of the granules, particularly noticeable over the characteristic discrete scrolls of T mast cells. T and TC mast cells each had large numbers of cytoplasmic granules, nuclei with peripherally condensed chromatin and low nuclear/cytoplasmic ratios, indicating maturity of both cell types. TC mast cell granules generally were more uniformly electron dense, larger and more numerous than T mast cell granules, which were more variable in shape. Compact solid-core scrolls, peripheral parallel lamellae and amorphous electron-dense material were found in granules of both cell types. Only TC mast cells had granules with grating and lattice substructures; only T mast cells had granules containing discrete scrolls. Less commonly, T mast cells were detected containing granules with a characteristic beaded or particulate ultrastructure. The ultrastructural features noted above were observed in T and TC mast cells regardless of the tissue in which they were examined and thereby permit T and TC mast cells to be distinguished by ultrastructure alone.

Distribution of meprin in kidneys from mice with high- and low-meprin activity.

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.

Deficiency of the tryptase-positive, chymase-negative mast cell type in gastrointestinal mucosa of patients with defective T lymphocyte function.

The distribution and concentration of human T (tryptase-positive, chymase-negative) and TC (tryptase-positive, chymase-positive) mast cells were examined in Carnoy's-fixed specimens of the gastrointestinal tract of normal individuals, patients with inflammatory bowel diseases, and patients with immunodeficiency disorders. In normal specimens, T mast cells predominated in the mucosa (89%), with a mean concentration of 17,850 +/- 4,998 per mm3 (+/- SD, n = 16), whereas TC mast cells predominated in the submucosa (90%) with a mean concentration of 7,516 +/- 1,227 per mm3 (+/- SD, n = 16). The concentrations of T and TC mast cells in specimens of ileum from five patients with active Crohn's disease and of colon from three patients with active ulcerative colitis were not significantly different (p greater than 0.4) from normal values. Three patients with combined immunodeficiency disorders demonstrated a marked decrease in the concentration of the T mast cells in the intestinal mucosa, to 540 +/- 630, and a corresponding decrease in the percentage of T mast cells to 9%. Concentrations of TC mast cells were unchanged, both in the mucosa and in the submucosa. In three patients with acquired immunodeficiency syndrome, a similar deficiency of the T mast cell type was observed in the ileal mucosa, with a mean concentration of 788 +/- 534 T mast cells per mm3, but not in the appendiceal and colonic mucosa of one of the three patients. These findings indicate a role for functional T lymphocytes in the development of the T mast cell type in humans, and suggest divergent pathways for development of T and TC mast cells.

Mast cells in human keloid, small intestine, and lung by an immunoperoxidase technique using a murine monoclonal antibody against tryptase.

A murine monoclonal antibody (G5) against human lung mast cell tryptase was used for selective staining of human mast cells by an indirect immunoperoxidase method. Human tissues (keloid, small bowel, lung) were fixed in either Carnoy's fluid or neutral buffered formalin. In all three tissues the number and location of G5-stained mast cells corresponded closely with metachromatic toluidine blue-stained mast cells, although the immunospecific technique appeared to be more sensitive. In lung the average concentration of G5-positive mast cells after Carnoy's fixation was 15,695/cu mm of subepithelial tissue in bronchi and bronchioles and 26,580/cu mm of alveolar wall, in small bowel was 20,958/cu mm of mucosa and 8576/cu mm of submucosa, and in keloid was 3068/cu mm. Formalin fixation significantly reduced concentrations of G5-positive mast cells in all tissues except keloid.