Heterogeneous ozone effects on the DNA methylome of bronchial cells observed in a crossover study.

We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.

Differential production of tumor necrosis factor, macrophage colony stimulating factor, and interleukin 1 by human alveolar macrophages.

Human alveolar macrophages (AMO) have been investigated for their ability to produce three monokines, tumor necrosis factor-alpha (TNF), macrophage colony stimulating factor (CSF-1), and interleukin 1-beta (IL-1). No TNF activity was found in supernatants of unstimulated AMO cultured for 20 h, although TNF mRNA was detected in the cells by Northern blot analysis. Stimulation of the cells with lipopolysaccharide (LPS) induced production and release of high levels of TNF into the culture supernatant. Increased levels of TNF mRNA were detectable at 90 min after LPS stimulation by dot blot analysis, reaching maximum expression between 4 and 8 h and declining thereafter. TNF activity peaked at approximately 8 h in the AMO supernatants. After 24 h TNF production had ended. Compared to autologous monocytes the AMO produced 5.7 times more TNF on a per cell basis (activity accumulated in 20 h supernatants). Uncultured AMO expressed CSF-1 mRNA which was translated into active protein recovered in supernatants upon culture in the absence of stimulus. The addition of LPS to AMO slightly reduced both mRNA levels and amount of factor in the supernatants. In contrast to the AMO, monocyte production of CSF-1 was enhanced by LPS. CSF-1 production by AMO continued for at least 48 h of culture. Spontaneous production of low amounts of IL-1 was found in one-third of the AMO samples, while low levels of IL-1 mRNA were present in all tested preparations. LPS stimulation induced increase in IL-1 mRNA within 90 min; mRNA levels peaked between 12 and 20 h and stayed high for at least 42 h. However, while all LPS-stimulated AMO expressed high levels of IL-1 mRNA biologically active IL-1 was again detected only in a fraction of the AMO supernatants. These results show that the production of monokines CSF-1, TNF, and IL-1 is differentially regulated by LPS in alveolar macrophages and has different responses as compared to monocytes. The longevity of the messages for each of the factors is possible indicators of the relative contribution of these factors to the response to endotoxin-induced injury and repair processes in the lung.

Identification of subpopulations that are sensitive to ozone exposure: use of end points currently available and potential use of laboratory-based end points under development.

A number of epidemiological studies have attempted to assess the effect of recurrent ozone exposure in humans. For the most part, they have failed to document convincingly an association between chronic ozone exposure and differences in lung function performance or respiratory symptoms. This is not surprising given the small respiratory effects observed in animals chronically exposed to ozone and assuming that people with abnormal respiratory function resulting from other occupational or environmental exposures, such as tobacco smoke, would make up a much larger percentage of the population than people with respiratory effects attributable to ozone. Therefore, either more sensitive end points must be developed to detect subtle changes due to chronic ozone exposure, or ways of selecting subpopulations that are especially sensitive to ozone must be devised. It has been well documented that there are large and reproducible differences in the acute response of individuals to ozone as measured by pulmonary function tests. Recently, it has also been shown that there are large differences in the acute response of individuals to ozone as measured by inflammatory and other biochemical parameters. This paper discusses the problems of selecting individuals who are sensitive to ozone depending on the end point chosen. It also describes potential new sensitive end points that might be available for ozone epidemiology studies in the near future.

Modulation of eicosanoid production by human alveolar macrophages exposed to silica in vitro.

Repeated inhalation of silica dust can lead to inflammation and fibrosis in human lung and in experimental animal models. The alveolar macrophage is believed to play a pivotal role in this process. Numerous macrophage-derived growth factors, cytokines, and arachidonic acid metabolites have been shown to contribute to inflammation and fibrosis. The objective of this study was to determine the eicosanoid production by human alveolar macrophages in response to silica exposure in vitro and to assess the contribution of alveolar macrophages to silica-induced fibrosis and inflammation. Macrophages were obtained from healthy volunteers and were incubated for 3 or 24 hr in the presence of silica (100, 60, and 0 micrograms/mL). Supernatants were removed for eicosanoid analysis. Eicosanoids were analyzed by both high performance liquid chromatography and radioimmunoassay. The data suggest that silica causes an increased release of leukotriene B4, leukotrienes C4/D4/E4, and 5-hydroxyeicosatetraenoic acid (5-HETE) after 3 hr and decreases in prostaglandin E2 and thromboxane B2 production after 24 hr of exposure to 100 micrograms/mL silica. In addition, 12-HETE and 15-HETE production remained unchanged at either time point. These opposing effects seen with the metabolites of lipoxygenase and cyclooxygenase pathways could contribute to silica-induced fibrosis. The pattern of eicosanoid production after exposure to silica was different from that obtained when macrophages were stimulated with lipopolysaccharide for 3 or 24 hr, indicating that the response to the particles was not just due to general cellular activation.

Inhalation of PM2.5 does not modulate host defense or immune parameters in blood or lung of normal human subjects.

We tested the hypothesis that exposure of healthy volunteers to concentrated ambient air particles (CAPS) between 0.1 and 2.5 microm in diameter is associated with modulation of human alveolar macrophage (AM) function, cytokine production, and immune phenotype in both blood and lung. Thirty-eight volunteers were exposed to either filtered air or CAPS from the immediate environment of the U.S. Environmental Protection Agency human studies facility in Chapel Hill, North Carolina, USA. Particle concentrations in the chamber during the exposures ranged from 23.1 to 311.1 microg/m3. No symptoms were noted by volunteers after the exposure. Eighteen hours after exposure, analysis of cells obtained by bronchoalveolar lavage (BAL) showed a mild increase in neutrophils in both the bronchial (8.4 +/- 2%) and alveolar fractions (4.2 +/- 1.7%) in subjects exposed to the highest concentration of CAPS compared to neutrophils in the fluids of those exposed to filtered air (bronchial fraction 2.7 +/- 0.6%; alveolar fraction 0.8 +/- 0.3%). There was no change in the percentage of lymphocytes or AMs recovered in the lavage after inhalation of the highest particle levels (mean 207 microg/m3). There was also no change in the proportion of lymphocytes in the BAL expressing CD3, CD4, CD8, CD19, nor activation markers CD25 or CD69. Particle inhalation did not affect the expression of CD11b, CD64, CD16, CD14, CD71 on AM, nor was there an effect on phagocytosis or oxidant generation following stimulation with zymosan A. IL-6 and IL-8 levels detected by enzyme-linked immunoabsorbent assay in the BAL were unrelated to inhaled particle levels. The distribution of lymphocyte subsets in blood obtained 18 hr after exposure to CAPS did not differ from that found before exposure. We conclude that ambient air particles are capable of inducing a mild inflammation in the lower respiratory tract but have no effect on immune phenotype or macrophage function under the conditions tested.

Do waste incinerators induce adverse respiratory effects? An air quality and epidemiological study of six communities.

The purpose of the study presented here was to simultaneously measure air quality and respiratory function and symptoms in populations living in the neighborhood of waste incinerators and to estimate the contribution of incinerator emissions to the particulate air mass in these neighborhoods. We studied the residents of three communities having, respectively, a biomedical and a municipal incinerator, and a liquid hazardous waste-burning industrial furnace. We compared results with three matched-comparison communities. We did not detect differences in concentrations of particulate matter among any of the three pairs of study communities. Average fine particulate (PM2.5) concentrations measured for 35 days varied across study communities from 16 to 32 micrograms/m3. Within the same community, daily concentrations of fine particulates varied by as much as eightfold, from 10 to 80 micrograms/m3, and were nearly identical within each pair of communities. Direct measurements of air quality and estimates based on a chemical mass balance receptor model showed that incinerator emissions did not have a major or even a modest impact on routinely monitored air pollutants. A onetime baseline descriptive survey (n = 6963) did not reveal consistent community differences in the prevalence of chronic or acute respiratory symptoms between incinerator and comparison communities, nor did we see a difference in baseline lung function tests or in the average peak expiratory flow rate measured over a period of 35 days. Based on this analysis of the first year of our study, we conclude that we have no evidence to reject the null hypothesis of no acute or chronic respiratory effects associated with residence in any of the three incinerator communities.

Human upper respiratory tract responses to inhaled pollutants with emphasis on nasal lavage.

A set of symptoms has been described during the past two decades. These symptoms, which have been called the sick building syndrome, include eye, nose, and throat irritation; headache; mental fatigue; and respiratory distress. It is likely that VOCs present in synthetic materials used in homes and office buildings contribute to these symptoms. There have been few studies, however, in which humans have been exposed to known amounts of VOCs under carefully controlled conditions. In this study, 14 subjects have been exposed to a mixture of VOCs (25 mg/m3 total hydrocarbon) representative of what is found in new homes and office buildings. Because irritation of the nose and throat are symptoms often associated with the upper respiratory tract and may result from an inflammatory response in the upper airways, we have used NAL to monitor PMN influx into the nasal passages following exposure to VOCs. We report statistically significant increases in PMNs both immediately after a 4-hr exposure to VOCs, as well as 18 hr later.

Canines as sentinel species for assessing chronic exposures to air pollutants: part 2. Cardiac pathology.

The principal objective of this study is to evaluate by light and electron microscopy (LM, EM) the heart tissues in stray southwest and northeast metropolitan Mexico City (SWMMC, NEMMC) dogs and compare their findings to those from 3 less polluted cities (Cuernavaca, Tlaxcala, and Tuxpam). Clinically healthy mongrel dogs, including 109 from highly polluted SWMMC and NEMMC, and 43 dogs from less polluted cities were studied. Dogs residing in cities with lower levels of pollutants showed little or no cardiac abnormalities. Mexico City and Cuernavaca dogs exhibited LM myocardial alterations including apoptotic myocytes, endothelial and immune effector cells, degranulated mast cells associated with scattered foci of mononuclear cells in left and right ventricles and interventricular septum, and clusters of adipocytes interspersed with mononuclear cells. Vascular changes included scattered polymorphonuclear leukocytes (PMN) margination and microthrombi in capillaries, and small venous and arteriolar blood vessels. Small veins exhibited smooth muscle cell hyperplasia, and arteriolar blood vessels showed deposition of particulate matter (PM) in the media and adventitia. Unmyelinated nerve fibers showed endoneural and epineural degranulated mast cells. EM examination of myocardial mast cells showed distended and abundant rough endoplasmic reticulum with few secretory granules. Myocardial capillaries exhibited fibrin deposition and their endothelial cells displayed increased luminal and abluminal pinocytic activity and the formation of anemone-like protrusions of the endothelium into the lumen. A close association between myocardial findings, lung epithelial and endothelial pathology, and chronic inflammatory lung changes was noted. The myocardial changes described in dogs exposed to ambient air pollutants may form the basis for developing hypothesis-driven mechanistic studies that might explain the epidemiological data of increased cardiovascular morbidity and mortality in people exposed to air pollutants.

Canines as sentinel species for assessing chronic exposures to air pollutants: part 1. Respiratory pathology.

A complex mixture of air pollutants is present in the ambient air in urban areas. People, animals, and vegetation are chronically and sequentially exposed to outdoor pollutants. The objective of this first of 2 studies is to evaluate by light and electron microscopy the lungs of Mexico City dogs and compare the results to those of 3 less polluted cities in MEXICO: One hundred fifty-two clinically healthy stray mongrel dogs (91 males/61 females), including 43 dogs from 3 less polluted cities, and 109 from southwest and northeast metropolitian Mexico City (SWMMC, NEMMC) were studied. Lungs of dogs living in Mexico City and Cuernavaca exhibited patchy chronic mononuclear cell infiltrates along with macrophages loaded with particulate matter (PM) surrounding the bronchiolar walls and extending into adjacent vascular structures; bronchiolar epithelial and smooth muscle hyperplasia, peribronchiolar fibrosis, microthrombi, and capillary and venule polymorphonuclear leukocytes (PMN) margination. Ultrafine PM was seen in alveolar type I and II cells, endothelial cells, interstitial macrophages (Mtheta), and intravascular Mtheta-like cells. Bronchoalveolar lavage showed significant numbers of alveolar macrophages undergoing proliferation. Exposure to complex mixtures of pollutants-predominantly particulate matter and ozone-is causing lung structural changes induced by the sustained inflammatory process and resulting in airway and vascular remodeling and altered repair. Cytokines released from both, circulating inflammatory and resident lung cells in response to endothelial and epithelial injury may be playing a role in the pathology described here. Deep concern exists for the potential of an increasing rise in lung diseases in child populations exposed to Mexico City's environment.

Neuropeptides and capsaicin stimulate the release of inflammatory cytokines in a human bronchial epithelial cell line.

The role of neuropeptides in initiating and modulating airway inflammation was examined in a human bronchial epithelial cell line (i.e. BEAS-2B). At a range of concentrations, exposure of BEAS-2B cells to Substance P (SP) or calcitonin gene related protein resulted in immediate increases in intracellular calcium ([Ca(2+)](i)), the synthesis of the transcripts for the inflammatory cytokines, IL-6, IL-8 and TNFalpha after 2 h exposure, and the release of their proteins after 6 h exposure. Addition of thiorphan (100 nM), an inhibitor of neutral endopeptidase, enhanced the levels of SP-stimulated cytokine release. Stimulation of IL-6 by SP occurred in a conventional receptor-mediated manner as demonstrated by its differential release by fragments SP 4-11 and SP 1-4 and by the blockage of IL-6 release with the non-peptide, NK-1 receptor antagonist, CP-99 994. In addition to the direct stimulation of inflammatory cytokines, SP (0.5 microM), in combination with TNFalpha (25 units/ml), synergistically stimulated IL-6 release. BEAS-2B cells also responded to the botanical irritant, capsaicin (10 microM) with increases in [Ca(2+)](i) and IL-8 cytokine release after 4 h exposure. The IL-8 release was dependent on the presence of extracellular calcium. Capsaicin-stimulated increases of [Ca(2+)](i) and cytokine release could be reduced to control levels by pre-exposure to capsazepine, an antagonist of capsaicin (i.e. vanilloid) receptor(s) or by deletion of extracellular calcium from the exposure media. The present data indicate that the BEAS-2B human epithelial cell line expresses neuropeptide and capsaicin-sensitive pathways, whose activation results in immediate increases of [Ca(2+)](i) stimulation of inflammatory cytokine transcripts and the release of their cytokine proteins.

2,3,7,8-TCDD induces cytochrome P450 enzyme activity but not proliferation or phenotypical changes in human peripheral blood lymphocytes.

In contrast to the well documented immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans accidentally or occupationally exposed to dioxin. More recently, a dose-dependent decrease of specific subpopulations of mitogen-activated human peripheral blood lymphocytes (PBL), including helper-inducer/memory cells (CD4+CD29+) and B cells (CD20+), was reported after in vitro treatment with dioxin concentrations as low as 10(-12)-10(-14) M TCDD [1]. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied in more detail, in order to assess the availability of a sensitive indicator system for human dioxin exposure. PBL from healthy volunteers were stimulated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (OKT3) and treated with 10(-7)-10(-14) M TCDD for 3-4 days. Cytochrome P450 (CYP1A1) enzyme induction was determined by the ethoxy-resorufin-O-deethylase (EROD) assay. Percentages of the different lymphocytes subsets, including CD2 (T cells), CD4, CD45 RA (suppressor-inducer/ virgin T cells), CD4, CD29, CD8, CD19 (B cells), as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (human leukocyte antigen HLA-DR) expression, were analyzed by flow cytometry. The proliferative activity was determined by 3H-thymidine uptake after 3-4 days of culture. In the present study, all stimulated lymphocyte cultures showed a significant increase of CYP1A1 activity at dioxin concentrations of 10(-7) and 10(-9) M. In contrast, alterations in surface antigen expression or suppression of proliferative responses did not occur in the mitogen-activated PBL over the whole concentration range of TCDD. These results clearly demonstrate that lymphoproliferation, as well as phenotypes of human PBL, are not affected by dioxin treatment and thus are not useful as sensitive biomarkers in human exposure studies.

Ozone effects on the immediate-phase response to allergen in the nasal airways of allergic asthmatic subjects.

Epidemiologic and clinical trials have suggested that exposure to ozone increases airway hyperresponsiveness and inflammatory response to inhaled nasal allergen challenge in allergic asthmatic subjects. Previous studies have demonstrated an increased late-phase response to nasal allergen challenge; however, the early-phase response is unknown. We sought to characterize the early-phase response by measuring mast-cell inflammatory mediators and cellular influx at time points immediately following ozone exposure and subsequent allergen challenge. A cohort of mild, asymptomatic dust mite--sensitive asthmatic subjects was identified. Each subject underwent two separate exposures to both 0.4 ppm ozone and clean air in a randomized manner. Nasal lavage was performed before and after each exposure. Nasal allergen was then administered to a defined clinical end point, followed by nasal lavage. Differential cell counts and mast-cell products were identified in each lavage specimen. The mast-cell mediators tryptase and prostaglandin D2 were analyzed, as was a marker of epithelial cell permeability, albumin. Although allergen produced an increase in early-onset mediator release (mast cell-derived), no enhancement was noted after exposure to ozone. Neutrophil and eosinophil inflammatory mediators were not increased after ozone exposure or enhanced after allergen exposure, although ozone did enhance eosinophilic influx after exposure to allergen. Ozone exposure does not promote early-phase--response mediator release or enhance the response to allergen challenge in the nasal airways of extrinsic asthmatic subjects. Ozone, however, may promote an inflammatory cell influx, which helps induce a more significant late-phase response in this population.

Respiratory tract pathology and cytokine imbalance in clinically healthy children chronically and sequentially exposed to air pollutants.

Chronic exposure of children to a complex mixture of air pollutants leads to recurrent episodes of upper and lower respiratory tract injury. An altered nasal mucociliary apparatus leaves the distal acinar airways more vulnerable to reactive gases and particulate matter (PM). The heterogeneity of structure in the human lung can impart significant variability in the distribution of ozone dose and particle deposition; this, in turn, influences the extent of epithelial injury and repair in chronically exposed children. Cytokines are low-molecular-weight proteins that act as intercellular mediators of inflammatory reactions, including lung injury of various etiologies. Cytokines are involved in generating inflammatory responses that contribute to injury at the lung epithelial and endothelial barriers. Mexico City is a 20-million-person megacity with severe air pollution problems. Southwest Metropolitan Mexico City (SWMMC) atmosphere is characterized by a complex mixture of air pollutants, including ozone, PM, and aldehydes. There is radiological evidence that significant lower respiratory tract damage is taking place in clinically healthy children chronically and sequentially exposed to air pollutants while growing up in SWMMC. We hypothesize that there is an imbalanced and dysregulated cytokine network in SWMMC children with overproduction of proinflammatory cytokines and cytokines involved in lung tissue repair and fibrosis. The nature of the sustained imbalance among the different cytokines ultimately determines the final lung histopathology, which would include subchronic inflammation, emphysema, and fibrosis. Cytokines likely would reach the systemic circulation and produce systemic effects. Individuals with an underlying respiratory or cardiovascular disease are less able to maintain equilibrium of the precarious cytokine networks.

Effect of ozone on susceptibility to respiratory viral infection and virus-induced cytokine secretion.

Airway epithelium is the primary target tissue for respiratory viruses as well as an important target of ozone (O(3)) toxicity. A change in the severity of viral airway infection may result from changes in epithelial cell susceptibility to infection, metabolic interference with viral replication, or altered production of immune regulatory molecules by the infected cells as a result of exposure to O(3). In this study we have investigated whether O(3) exposure alters the susceptibility of human airway epithelial cells to respiratory syncytial virus (RSV) infection, the production of infectious virus, and/or release of virus-induced cytokines IL-6 and IL-8. The epithelial cell line BEAS-2B grown on collagen-impregnated filters was exposed to O(3) (0.5 ppm for 60 min) or filtered air immediately before or 24 h after infection with RSV. Cells exposed to O(3) before RSV infection released 44% less virus over 4 days of infection while O(3) exposure post RSV infection had no effect on virus production. O(3) exposure preceding RSV infection showed short term additive effects of these treatments on epithelial cell IL-6 and IL-8 production, a decrease in cytokines at 48 h, but did not affect long term cytokine production by RSV-infected cells. Furthermore, O(3) exposure did not affect long term cytokine production by cells with an established RSV infection at the time of exposure. These data suggest that O(3) does not adversely affect viral airway infection, at least not on the level of the host cell for viral replication.

Exposure of humans to a volatile organic mixture. III. Inflammatory response.

A set of symptoms has been described during the past two decades that has been called the "sick building syndrome." These symptoms include eye, nose, and throat irritation; headache; mental fatigue; and respiratory distress. It is likely that the volatile organic compounds (VOCs) present in synthetic materials used in homes and office buildings contribute to these symptoms. However, there have been very few studies in which humans have been exposed to known amounts of VOCs under carefully controlled conditions. In this study, 14 subjects were exposed to a mixture of VOCs (25 mg/m3 total hydrocarbon) that is representative of what is found in new homes and office buildings. Because irritations of the nose and throat are symptoms often associated with the upper respiratory tract and may result from an inflammatory response in the upper airways, we used nasal lavage to monitor neutrophil (PMN) influx into the nasal passages following exposure to VOCs. There were statistically significant increases in PMNs, both immediately after a 4-h exposure to VOCs and 18 h later.

Respiratory response of humans exposed to low levels of ozone for 6.6 hours.

Recent evidence suggests that prolonged exposures of exercising men to 0.08 ppm ozone (O3) result in significant decrements in lung function, induction of respiratory symptoms, and increases in nonspecific airway reactivity. The purpose of this study was to confirm or refute these findings by exposing 38 healthy young men to 0.08 ppm O3 for 6.6 h. During exposure, subjects performed exercise for a total of 5 h, which required a minute ventilation of 40 l/min. Significant O3-induced decrements were observed for forced vital capacity (FVC, -0.25 l), forced expiratory volume in 1 s (FEV1.0, -0.35 l), and mean expiratory flow rate between 25% and 75% of FVC (FEF25-75, -0.57 l/s), and significant increases were observed in airway reactivity (35%), specific airway resistance (0.77 cm H2O/s), and respiratory symptoms. These results essentially confirm previous findings. A large range in individual responses was noted (e.g., percentage change in FEV1.0; 4% increase to 38% decrease). Responses also appeared to be nonlinear in time under these experimental conditions.

Biogenesis of the mitochondrial ATPase from sea urchin embryos.

The mitochondrial rutamycin-sensitive ATPase from sea urchin eggs was purified to homogeneity. The subunit structure of the enzyme was characterized by SDS-gel electrophoresis. Eight polypeptides were identified with molecular weights of 55,000, 52,000, 39,000, 31,000, 28,000, 23,000, 17,000 and 10,000. Developing sea urchin embryos were incubated with [2H]leucine in the presence of emetine preferentially to label mitochondrially made proteins. Under these conditions sea urchin mitochondria synthesize eight different polypeptides. Two of these proteins, with molecular weights of 31,000 and 23,000, co-purify with the ATPase. Antibody directed against the pure rutamycin-sensitive ATPase precipitated only these two proteins. Therefore, two of the eight sea urchin ATPase subunits appear to be made by mitochondria.

Coordinate regulation of contractile protein synthesis during myoblast differentiation.

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.

Use of a cDNA library for the study of mRNA changes during muscle differentiation.

A cDNA library was used to measure changes in many individual mRNAs during muscle differentiation in culture. A library of 1000 clones was constructed from total myofiber poly(A) RNA. About 23% of these clones gave a detectable colony hybridization signal using end-labeled myofiber mRNA, the remainder containing muscle sequences too rare to be detected with this assay. The 230 positive clones were grouped into four classes based on relative visual intensity. Reconstruction experiments using pure globin mRNA enable us to determine the approximate percentage of total RNA made up by each mRNA hybridizing to a cDNA clone. Those clones containing sequences complementary to developmentally regulated mRNAs were identified by a differential hybridization procedure. The cDNA library was screened with end-labeled mRNA from both undifferentiated myoblasts and differentiated myofibers. Although the bulk of the clones hybridized essentially the same with both RNA populations, several dozen were found which hybridized differentially. Some clones contained sequences which were not present at all in myoblasts and present in very high quantities in myofibers. Others contained sequences found in both myoblasts and myofibers but in increased quantities in the differentiated cells. Still others contained sequences which decreased in quantity during muscle differentiation. The clones in the first group were chosen for immediate analysis since they likely contain contractile protein mRNA sequences. However, all the characterized cDNA clones can now be used as probes to study the chromosomal organization and developmental expression of genes active during muscle differentiation.