Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle

Sclerosis and reduced microvessel density characterize advanced stages of muscular dystrophy and hamper cell or gene delivery, precluding treatment of most individuals with Duchenne muscular dystrophy. Modified tendon fibroblasts expressing an angiogenic factor (placenta growth factor, PlGF) and a metalloproteinase (matrix metalloproteinase-9, MMP-9) are able to restore a vascular network and reduce collagen deposition, allowing efficient cell therapy in aged dystrophic mice. These data open the possibility of extending new therapies to currently untreatable individuals.     

Human DNA replication: Fiber autoradiographic analysis of diploid cells from normal adults and from Fanconi's anemia and ataxia telangiectasia

Summary Fiber autoradiograms prepared from radioactive DNA of diploid skin fibroblasts and lymphocytes from normal adult humans show patterns of replication of chromosomal DNA that are closely similar to those from other mammalian cells. The rate of replication fork movement is 0.64 m/min and 0.42 m/min for fibroblasts and lymphocytes respectively. In both types of cells, replication units show a median length of about 70 m. Replication proceeds bidirectionally from a central origin in most units and adjacents units initiate synthesis synchronously. In skin fibroblasts from individuals with Fanconi's anemia or ataxia telangiectasia, the autoradiographic patterns of DNA replication are the same as in controls. This suggests that S phase DNA synthesis is normal in these disorders.     

Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications

Summary The distribution and frequency of aphidicolin-induced common fragile sites were studied in chromosomes of cultured skin fibroblasts and PHA-stimulated lymphocytes from five normal individuals; 0.2 M aphidicolin was added for the last 26 h of culture. Skin fibroblasts from five fra(X)-positive patients were also studied in the same manner. Fragile sites most frequently found in fibroblasts from normal individuals were 3q26.2, 7q11.23, 16q23, 1p31, 10q11.2, 12q23 and 7q31, whereas those in lymphocytes from the same individuals were 3p14, 16q23, Xp22, 7q32 and 14q24. The distribution of fragile sites in fibroblasts from fra(X)-positive patients was essentially identical with that in normal individuals. The average number of gaps and breaks in 100 metaphases was 36.8 in fibroblasts from normal individuals, 113.8 in those from fra(X)-positive patients, and 279 in lymphocytes from normal individuals. Their rates of chromosome-type breaks and gaps were 7.9%, 29.7% and 54.5%, respectively. Thus, the distribution and frequency of aphidicolin-induced fragile sites were different between skin fibroblasts and lymphocytes, possibly reflecting differences in their DNA replication sequence or gene activity.     

Molecular cytogenetic study of patients with Pallister-Killian syndrome

The Pallister-Killian syndrome (PKS) is characterized by tissue limited chromosomal mosaicism, i.e. the presence of a supernumerary metacentric chromosome [i(12p)] often confined to skin fibroblasts while the karyotype of cultured lymphocytes is normal. In the present study, chromosome painting by chromosomal in situ suppression (CISS) hybridization and interphase cytogenetic procedures employing biotinylated or digoxigenin labelled probes was carried out. These probes comprised a chromosome 12 specific library (LA 12NSO1) and chromosome 12 centromere specific -satellite (pSP12-1). They were used to analyse and quantify the presence of i(12p) in lymphocytes, granulocytes/monocytes, skin fibroblasts and buccal mucosal cells from five patients and one aborted fetus with PKS, and ten normal donors. CISS hybridization on mitotic skin fibroblasts reliably indicated the presence of i(12p) cells, even when metaphases of poor quality were included in the analysis. Two of the five patients showed i(12p) in a small proportion (0.5%) of the cultured lymphocytes too. The interphase cytogenetics procedure did not reveal the isochromosome in lymphocytes or granulocytes/monocytes in any of the patients. Two of the six patients had a twofold increase in the number of buccal mucosal cells with three hybridization signals over control values. However, for mucosal cells, methodological improvements are required. For cytogenetic diagnosis of PKS, cultured fibroblasts subjected to chromosome painting by CISS hybridization with a chromosome 12 specific library probe are recommended.     

Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry

An important intention of flow cytometric investigations is to obtain biochemical and biophysical information about cells which is suitable for automated tumor diagnosis. In this study, the ploidy status, the intracellular pH value, the intracellular esterase activity, and the cell volume of vital cells and the DNA and cell volume of dead cells were measured in cancerous tissue and normal lung tissue of 30 patients by flow cytometry. The cell samples were simultaneously stained with the pH and esterase indicator dye 1.4-diacetoxy-2,3-dicyanobenzene (ADB) and propidium iodide (PI). The flow cytometric measurements were performed in three-parameter list mode. The data were evaluated on an AT-compatible personal computer with the DIAGNOS1 program system for automated diagnosis of flow cytometric list mode data. Significant differences were found between normal and malignant tissue in DNA ploidy, in the intracellular esterase activity, in the cell, volume and in the percentage of inflammatory cells and parameters of necrosis. DNA-aneuploidy was observed in 38% of the lung carcinomas. The simultaneous detection of DNA-aneuploidy and tumor-associated properties in a multifactorial analysis led to correct automatic tumor diagnosis in 85% of cases. DNA-aneuploidy was found at a significant higher frequency in advanced tumors. Adenocarcinomas displayed DNA-aneuploidy more often (80%) than squamous cell carcinomas (33%).     

GSH system in relation to redox state in dystrophic skin fibroblasts

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

<Emphasis Type="Italic">LARGE</Emphasis> expression in different types of muscular dystrophies other than dystroglycanopathy

Background

Alpha-dystroglycan (αDG) is an extracellular peripheral glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin globular domains and certain arenaviruses. An important enzyme, known as Like-acetylglucosaminyltransferase (LARGE), has been shown to transfer repeating units of -glucuronic acid-β1,3-xylose-α1,3- (matriglycan) to αDG that is required for functional receptor as an extracellular matrix protein scaffold. The reduction in the amount of LARGE-dependent matriglycan result in heterogeneous forms of dystroglycanopathy that is associated with hypoglycosylation of αDG and a consequent lack of ligand-binding activity. Our aim was to investigate whether LARGE expression showed correlation with glycosylation of αDG and histopathological parameters in different types of muscular dystrophies, except for dystroglycanopathies.

Methods

The expression level of LARGE and glycosylation status of αDG were examined in skeletal muscle biopsies from 26 patients with various forms of muscular dystrophy [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), sarcoglycanopathy, dysferlinopathy, calpainopathy, and merosin and collagen VI deficient congenital muscular dystrophies (CMDs)] and correlation of results with different histopathological features was investigated.

Results

Despite the fact that these diseases are not caused by defects of glycosyltransferases, decreased expression of LARGE was detected in many patient samples, partly correlating with the type of muscular dystrophy. Although immunolabelling of fully glycosylated αDG with VIA4–1 was reduced in dystrophinopathy patients, no significant relationship between reduction of LARGE expression and αDG hypoglycosylation was detected. Also, Merosin deficient CMD patients showed normal immunostaining with αDG despite severe reduction of LARGE expression.

Conclusions

Our data shows that it is not always possible to correlate LARGE expression and αDG glycosylation in different types of muscular dystrophies and suggests that there might be differences in αDG processing by LARGE which could be regulated under different pathological conditions.

     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

Membrane Repair Defects in Muscular Dystrophy Are Linked to Altered Interaction between MG53, Caveolin-3, and Dysferlin

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis and maintenance of sarcolemma integrity in striated muscle. During the life cycle of striated muscle, membrane repair is a fundamental process in maintaining cellular integrity, as shown by recent studies that link defective membrane repair to the progression of muscular dystrophy (1–3). Repair of the plasma membrane damage requires recruitment of intracellular vesicles to injury sites (4, 5). One protein that has been linked to membrane repair in skeletal muscle is dysferlin (6, 7), which is thought to fuse intracellular vesicles to patch the damaged membrane and restore sarcolemmal integrity following muscle injury. Like dysferlin, caveolin-3 (Cav3)3 is a muscle-specific protein, and many mutations in Cav3, including P104L, R26Q, and C71W, have been linked to muscular dystrophy (8–11). Despite extensive research efforts on Cav3 and dysferlin (12–14), the molecular function of these two proteins in membrane repair in muscle physiology and dystrophy have not been fully defined.Animal model studies reveal that either loss or gain of Cav3 function both result in dystrophic phenotypes in skeletal muscle (15, 16), suggesting that associated cellular components may be involved in the etiology of Cav3-related dystrophy. Although the discovery of dysferlin highlights the importance of membrane repair in the etiology of muscular dystrophy, dysferlin itself does not appear to participate in recruitment of intracellular vesicles because dysferlin^−/− muscle retains accumulation of vesicles near membrane damage sites (7). This indicates that proteins other than dysferlin are required for nucleation of intracellular vesicles at the sites of acute membrane damage. Recently, we discovered that MG53, a muscle-specific TRIM family protein (TRIM72), is an essential component of the acute membrane repair machinery. MG53 acts as a sensor of oxidation to nucleate recruitment of intracellular vesicles to the injury site for membrane patch formation (17). We also found that MG53 can regulate membrane budding and exocytosis in muscle cells, and this membrane-recycling function of MG53 can be modulated through a functional interaction with Cav3 (18).Here we present evidence that MG53 interacts with dysferlin to facilitate intracellular vesicle trafficking during repair of acute membrane damage. In addition, we show that transgenic overexpression of P104L-Cav3 in striated muscle produces defects in membrane repair that are linked to altered subcellular distribution of MG53 and dysferlin. Our results suggest that altered MG53 localization can be used as a marker for muscular dystrophy involving reduced sarcolemmal membrane repair capacity due to Cav3 mutation, and potentially, in other forms of dystrophy as well.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls

The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33 000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist. The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell-wall fragments are pre-incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre-incubation. This implies, as postulated from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, delta psi, at the cell surface. The cell-wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell-wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co-operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell-wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell-wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active. As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum. The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co-operative with respect to pH.     

Behaviour of Duchenne dystrophy fibroblasts in collagen gels

In order to clarify the possible involvement of the cell surface in the pathogenesis of Duchenne muscular dystrophy, we have examined the behaviour of fibroblasts cultured from Duchenne patients in hydrated collagen lattices. No differences could be found between Duchenne and normal skin fibroblasts, either after initial seeding or following prolonged culture within the collagen gel.     

Expression of an X-linked muscular dystrophy in a female due to translocation involving Xp21 and non-random inactivation of the normal X chromosome

Summary A young female was diagnosed as having X-linked muscular dystrophy of the Duchenne type. Chromosome studies, including trypsin-Giemsa banding, Quinacrine fluorescence, and nucleolus organizer region (NOR) silver staining revealed an X-autosome reciprocal translocation t(X;21) (p21;p12). Utilizing both [³H] thymidine autoradiography and the BrdU-Hoechst 33258-Giemsa technique, lymphocytes and fibroblasts were found to show a preferential inactivation of the normal X suggesting the presence of a single mutant gene on the translocated X. This patient is one of seven reported cases of an X-linked muscular dystrophy associated with an X-autosome translocation. In all seven cases the exchange point in the X chromosome is in band p21 at or near the site of the Duchenne gene.     

Volume-stimulated,Cl−-dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS

Summary We report here that a Cl^–-dependent K^– (KCl) efflux, which is stimulated by N-ethylmaleimide, (NEM) and by increased red cell volume, exists in young red cells of individuals with normal hemoglobin A (AA) and in those homozygous for hemoglobin S (SS). We have investigated this KCl efflux in several density-defined red cell fractions obtained from Percoll-Stractan continuous density gradients. We found high activity of the NEM-stimulated KCl transport in reticulocytes and young red cells from nine sickle cell (SS) patients (43±27 mean±sd mmol K⁺/liter of cells/hr=flux units (FU)) and in the young cell fraction of three AA individuals with high reticulocytosis recuperating from nutritional anemias (41.7±10 FU). In addition, we observed significant interindividual variation of this KCl efflux in the discocyte fraction of SS blood. Cell swelling markedly stimulated the KCl efflux, in SS whole blood (9.8±7.4 FU, in SS young cells (13±13 FU), and in AA young cells (21.4±11 FU). The activity of the Na–K–Cl cotransport, as estimated by the bumetanide sensitive K⁺ efflux was not found to be cell-age dependent in either AA or SS cells.Measurements of red cell density by isopycnic gradients indicated that 27% of the young cells reduce their volume by a Cl^–-dependent process in hypotonic or low pH-induced swelling.The large volume-stimulated KCl efflux in AA young cells raises the possibility that these fluxes may be involved in the maturation of erythropoietic precursors. The high activity in the red cells of sickle cell anemia patients and its interindividual variation may have pathophysiological consequences since it reverses the decrease in the intracellular concentration of hemoglobin which occurs in response to low pH or osmolarity, an unwelcome pro-sickling event.     

The differential effects of calcium starvation on Duchenne muscular dystrophy fibroblasts

The effects of nutrient deprivation on normal and Duchenne muscular dystrophy fibroblasts were examined. The requirements for Ca2+ and fetal bovine serum were assessed by their effects on the cells' ability to support viral replication, and by ability of the cells to divide in the presence of low levels of these nutrients. When grown in Ca2+-deficient media, Duchenne fibroblasts supported viral replication at a rate 2- to 2.5-fold greater than did normal fibroblasts. At normal Ca2+ levels, Duchenne fibroblasts supported viral replication at levels slightly lower than their normal counterparts. After 48 hr in medium containing 0.2 mM Ca2+, the growth of normal cells was arrested, while Duchenne fibroblasts were relatively unaffected. When grown in medium containing either 0.2 or 2.0% serum, the growth of normal cells was arrested within 48 hr, with cell death occurring within 72 hr. Duchenne fibroblasts continued to divide at these serum levels for 72 hr, reaching higher cell densities than normal cells. These results suggest that a defect related to Ca2+ metabolism may be part of the Duchenne phenotype, which could be used to identify Duchenne muscular dystrophy cells.     

Rotational relaxation time of concanavalin A bound to the surface membrane of normal and malignant transformed cells

Fluorescence polarization was used to determine the rotational relaxation time of fluorescein conjugated concanavalin A bound to the surface membrane of normal and malignant cells. The cells used were normal lymphocytes and malignant lymphoma cells, as examples of cells that are in suspension in vivo, and normal and simian virus 40-transformed fibroblasts, as examples of cells that form a solid tissue. The relaxation time of F-concanavalin A² in phosphate-buffered saline, pH 7.2, at 24 °C was 58 nseconds. Under the same conditions, the relaxation times of F-concanavalin A bound to cells were: 70 nseconds for normal lymphocytes, 160 nseconds for malignant lymphoma cells, 120 nseconds for normal fibroblasts and 73 nseconds for simian virus 40-transformed fibroblasts. When cells were treated with trypsin or glutaraldehyde before adding F-concanavalin A, trypsin treatment produced a decrease, whereas glutaraldehyde fixation produced an increase of these values. Inhibition of cap formation of concanavalin A binding sites on normal lymphocytes by treating the cells with sodium azide did not change the rotational relaxation time of concanavalin A bound to the cells. These results indicate that the carbohydrate-containing structures on the cells that bind concanavalin A are mobile. In cells that are in suspension in vivo, malignant transformation is associated with reduction in mobility of these sites. However, in cells that form a solid tissue, malignant transformation is associated with an increase in mobility of these sites. Determination of the rotational relaxation time of fluorescent probes bound to specific sites on cell membranes can thus be used to quantitate receptor mobility in relation to cell behaviour.     

5'-Nucleotidase in skin fibroblasts from patients with Duchenne muscular dystrophy

The 5'-nucleotidase of plasma membranes of cultured skin fibroblasts from patients with Duchenne muscular dystrophy had a reduced affinity for its substrate, 5'-AMP. The Arrhenius plot of the temperature dependence of this enzyme activity was normal. There was no difference between patients and controls in the specific 5'-nucleotidase activity in the whole cell homogenates.