The distribution of anionic sites on the surface of the Golgi complex.

The distribution of anionic binding sites has been investigated in the isolated Golgi complex using cationic ferritin. The greatest density of anionic sites occurs on the tubular network and small vesicles, and this binding is accompanied by increased levels of galactosyltransferase activity. The density of anionic sites on the cisternae is less than on the tubules and shows anisotropic distribution, with higher density on the convex surface and lower density on the concave surface. The distribution of anionic sites may reflect the functional activity of the Golgi complex and possibly the interaction or cohesion between cisternae in this organelle.

The effect of temperature on the galactosyl- and sialyltransferases and on the ultrastructure of Golgi membranes.

The effect of temperature on the activity of galactosyl- and sialyltransferases of rat liver Golgi membranes and the galactosyltransferase of serum has been studied. Arrhenius plots for three enzymes were different. Sharp breaks in the curves, indicative of phase transitions were observed for sialyltransferase (28 degree C) of Golgi and galactosyltransferase (34 degree C) of serum but not for galactosyltransferase of Golgi. The activation energy was greater above the break (above 28 degree C) than below for sialyltransferase of Golgi; The activation energy was lower (above 34 degree C) for galactosyltransferase of serum than below. Electron microscopic freeze replicas showed a patchy distribution of particles which increased as the temperature was raised accompanied by smooth areas. This was interpreted as representing lateral phase separation of the membrane components.

The effect of cycloheximide on galactosyltransferase of rat liver Golgi membranes.

An inhibitory effect of cycloheximide on the initial rate of galactosyltransferase of rat liver Golgi membranes has been demonstrated. Cycloheximide was effective in inhibiting the activity of the enzyme when added directly to the assay medium or after pre-incubation of the membranes with the drug. The inhibition observed with different concentrations of nucleotide sugar was shown to be competitive at higher concentrations of the nucleotide sugar (0.10-1.0 mumol). The inhibition observed with different concentrations of acceptor, N-acetylglucosamine was complex and could not be analysed further with the present data. Washing the Golgi membranes previously incubated with cycloheximide with water failed to reverse the inhibition. Washing with UDPgalactose partially reversed the inhibition only. These results, together with the observation that serum galactosyltransferase was not inhibited by cycloheximide supported the view that the cycloheximide effect may be primarily on the membrane system.

Cystic fibrosis in Ontario.

The incidence of cystic fibrosis in Ontario, Canada has been determined from clinical data, from the cystic fibrosis database of the Hospital for Sick Children, Toronto, and from population statistics in the Province of Ontario. The survey included 420 confirmed cases of cystic fibrosis born during the period 1966-1980. The mean incidence during this period was one in 2,927. In the last 5-year period, a decline was noted in incidence that may have reflected in part the effectiveness of early diagnosis and genetic counseling in affected families. During the period of the survey, over 60% of cases were diagnosed within the first year of life, 74% by age 2 years, and 90% by age 5 years. Clinical diagnosis in the first year of life was more common in males (65%) than in females (54%), a consistent finding during the period of the survey. The incidence of meconium ileus was 15.7% of ascertained cases of cystic fibrosis, with similar incidences in males (16.4%) and females (14.4%). Although survival has not been the subject of this survey, mortality in the neonatal period was significantly higher in males than in females with cystic fibrosis.

Genetic aspects of immotile cilia syndrome.

The genetics of the immotile cilia syndrome has been analyzed in a series of 46 affected individuals from 38 families. Both sexes were equally affected: there were 20 males and 26 females in this series. All patients had upper and lower respiratory disease with chronic sinusitis, otitis, and chronic cough from early childhood. Bronchiectasis was common in older children and adults. Situs inversus occurred randomly, affecting 11 males and 15 females. Biopsies of nasal and bronchial mucosa from these subjects have been investigated by electron microscopy and identified as having specific ultrastructural defects of respiratory tract cilia including deficiencies in outer dynein arms (19), inner dynein arms (3), both inner and outer dynein arms (15), radial spoke defect (5); and microtubular transposition anomaly (4). Segregation analysis of proband sibships was consistent with autosomal recessive inheritance. However, the different ultrastructural defects that underly the immotile cilia syndrome involve presumably different genetic determinants, and the different types have not been analyzed separately. Examination of paternal age and birth order gave no evidence of new autosomal dominant mutation in the series.

Genital mycoplasma infection. Intrauterine infection: pathologic study of the fetus and placenta.

Mycoplasma infection was present in the fetuses from three spontaneous abortions and in one second-trimester newborn. Gross examination revealed in most cases a severely infected placenta and membranes, with a fetus of normal appearance. The fetal infection presumably followed placental involvement and appeared to have been acquired shortly prior to delivery. Genital mycoplasmas, Ureaplasma urealyticum or Mycoplasma hominis, were isolated from the placentas and the fetal tissues, and from the genital tracts of the mothers. Isolation of mycoplasmas from the liver indicated that bloodstream dissemination of these organisms occurred in the fetus. In the fetus, the pathologic changes were variable. Lesions were identified in the lung by scanning electron microscopy of the bronchial tree in two cases and were accompanied by interstitial pneumonia. An abnormally dilated left ventricle suggestive of cardiomyopathy was observed in one case.

Parkinson-like syndrome in nonhuman primates receiving a tetrahydropyridine derivative.

Antipsychotic drugs, while ameliorating symptoms in schizophrenia, evoke extrapyramidal effects which resemble parkinsonism. We studied the potential of 1- (4,4-bis(4-fluorophenyl)butyl)-4-(4-fluorophenoxy)-1,2,3,6-tetrahydropyr idine d-tartrate to induce extrapyramidal side effects in Rhesus monkeys. This agent shares neurochemical effects of known antipsychotic agents in its ability to antagonize cerebral dopamine action by competing for (3H)-Haloperidol binding of the dopamine receptors and inhibiting limbic and striatal adenylate cyclase in rat brain. The compound was administered orally to monkeys for 18 days, starting at 2 mg/kg and increasing to 20 mg/kg. Additional groups of monkeys received the drug for 29 consecutive days at 5 and 7.5 mg/kg/day. In both studies, extrapyramidal signs were associated with neuropathological changes in the brains of treated monkeys. The findings resemble those reported in Rhesus monkeys and in drug addicts after repeated intravenous administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The findings also suggest a structure/activity relationship of tetrahydropyridine analogs with neurologic and associated neuropathologic manifestations produced in monkeys. The experimental model is useful to study the pathogenesis and possibly therapeutic approaches for Parkinson's disease.

The Golgi complex. III. The effects of puromycin on ultrastructure and glycoprotein synthesis.

The effect of puromycin has been investigated on protein and glycoprotein synthesis and on ultrastructure of the Golgi complex from rat liver. Incorporation of [14C]leucine into protein in Golgi fractions and into serum proteins was depressed rapidly after puromycin treatment. In the serum proteins, incorporation returned to normal levels at 2 h whereas in Golgi fractions it continued to rise to 200% of the control levels at 3 h and was still elevated at 24 h after puromycin treatment. Incorporation of [14C]glucosamine into glycoprotein was depressed in Golgi and serum fractions in a similar manner but slightly later than that of leucine. Leucine labelled material found at 3 h was a poor acceptor for carbohydrate, since [14C]glucosamine incorporation was not elevated above control values. Galactosyl transferase activity was not depressed in the Golgi membranes and, at 3 h, was elevated implying that an adequate supply of enzyme was available at all times. The activity of the galactosyl transferase in serum appeared to be depressed suggesting that transport of enzyme from Golgi complex to serum was defective. Ultrastructural changes in the Golgi complex were observed to occur rapidly after puromycin treatment. The cisternae became irregular, compressed, and degenerated progressively from central region towards the periphery. Irregular tubular structures formed at the expense of cisternal membrane and showed accumulation of low density lipoprotein. Vesiculation and degenerative changes of the Golgi membranes continued from 2-12 h while more typical arrangements of the Golgi complex were observed between 24-48 h. The morphological changes correlated with changes in glycoprotein synthesis.

The secretory response in dissociated acini from the rat submandibular gland.

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to beta-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of 14C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range t x 10(-5) to 5x 10(-7) M and to 110% of controls at 5 x 10(-8) M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 x 10(-5) M and to 115% at 5 x 10(-6) M but had no effect at 5 x 10(-7) or 5 x 10(-8) M. The secretory response was blocked by the respective beta-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.

Histiocytic sarcoma in Wistar rats. A light microscopic, immunohistochemical, and ultrastructural study.

Histiocytic sarcoma, a recently described tumor entity in rats, was studied by light microscopy in 20 male and female Wistar rats. The tumors originated from subcutaneous tissues; metastasis involved primarily the liver with sinusoidal spread and the lungs with peribronchiolar distribution. The characteristic features of this tumor were the uniform population of tumor cells, palisading necrosis, and abundant multinucleated giant cells. Immunocytochemical and ultrastructural findings confirmed the histiocytic nature of the tumor cells.

Abnormalities of cilia in the middle ear in chronic otitis media.

We studied the fine structure of nasal and middle ear mucosa in 18 patients with chronic otitis media, comparing them with ten age-matched control patients. Electron microscopy revealed no single fundamental defect of cilia in the tissue samples; however, patients with chronic otitis media had a significantly higher incidence of abnormal cilia in the middle ear without correlating abnormalities of nasal cilia. These findings indicated that ciliary anomalies, including deletion of ciliary microtubules, presence of supernumerary tubules, and fusion of cilia, occur as a result of infection or inflammation. Such abnormalities would impair normal mucociliary clearance and exacerbate clinical complications.

Structural and developmental abnormalities of the exocrine pancreas in cystic fibrosis.

Normal infants from 32 to 54 weeks post-conceptional age show a clear pathway of development in the exocrine pancreas, involving differentiation of acinar tissue, reflected by increased acinar to connective tissue volumes. In contrast, an abnormal pattern of development has been found in cystic fibrosis. Early signs of a deficiency in exocrine tissue at 32-38 weeks post-conceptional age suggest that there is a lack of normal maturation of pancreatic exocrine tissue that occurs in utero, with a degenerative process supervening after birth. The volumes of the acinar and duct lumen is markedly increased, up to 10 fold normal volume in cystic fibrosis subjects. However, the lumenal changes are not a function of age. Accumulation of secretory material in the duct is a characteristic feature in cystic fibrosis and may cause dilation of the duct and acinar lumen and degradation of acini. Alternatively, the greater volume and lack of direct relationship to exocrine acinar volume may reflect a persistence of the fetal pattern of pancreatic development in cystic fibrosis. The diagnosis of cystic fibrosis within the first few months of life is difficult when based on conventional or subjective histologic criteria. By quantitative microscopy of the pancreas, an objective approach is available that clearly distinguishes cystic fibrosis from control subjects. In this retrospective survey, 93% of cystic fibrosis infants were discriminated from normals; only 2 of 30 cases (70%) were not clearly differentiated from controls. As a genetic disorder, the manifestation of cystic fibrosis would be expected to result in pancreatic dysfunction in utero. Other predisposing factors, however, may be involved in the pancreatic lesions such as obstruction of the pancreatic duct, failure in the synthesis or secretion of pancreatic enzymes or abnormal mucus production in the intestine. Further ultrastructural and functional investigations will be important to understand the underlying defect in cystic fibrosis.

The mucous lining of major bronchi in the rabbit lung.

Mucous secretions covering the surface of the trachea and major bronchi of the rabbit lung have been demonstrated with the scanning electron microscope. The surface secretion forms a continuous layer overlying the major airways and shows a complex 3-dimensional arrangement of overlapping sheets and fibrous networks. In the main and lobar bronchi, mucus forms more expanded networks, and in the trachea, mucus forms a smoother, more cohesive layer overlying the epithelial cells. The mucous blanket follows closely the contours of the airway epithelium and is situated at the tips of the cilia.

Hydrogen peroxide release by rat alveolar macrophages: comparison with blood neutrophils.

Hydrogen peroxide release was examined using biochemical and cytochemical techniques in rat alveolar macrophages, at rest and during phagocytosis, and compared with rat blood neutrophils. Using biochemical techniques, alveolar macrophages released small amounts of hydrogen peroxide at rest, and no increase was observed after challenge with opsonized and nonopsonized zymosan particles at several particle-cell ratios (1:1 to 1:1,000). Neutrophils released similar quantities of hydrogen peroxide at rest but showed a 12-fold increase in hydrogen peroxide release following exposure to opsonized zymosan particles. Using cytochemical techniques to localize sites of hydrogen peroxide release, resting neutrophils showed little deposition of reaction product at the cell surface and occasional deposits in endocytotic vesicles. After exposure to latex particles, a dense reaction product was observed between the particle and the cell membrane, indicating significant increases in hydrogen peroxide release at the sites of particle contact with the neutrophil. The resting macrophage displayed a light, uniform precipitation of cerium over the cell surface and lining intracellular channels and endocytotic vesicles and vacuoles. Following particle exposure, there was no significant difference in the density or distribution of reaction product. These findings, together with previous studies of oxidative metabolism, suggest that alveolar macrophages do not release increased quantities of hydrogen peroxide during phagocytosis. In contrast to neutrophils, oxidative-dependent metabolic pathways may not be of primary importance for microbial killing by alveolar macrophages.

Cystic fibrosis: effects of serum factors on mucus secretion.

The effects of serum from children with cystic fibrosis and from normal children on the mucus-secreting, ciliated epithelium have been investigated in vitro using explanted tissue from rabbit lung. By optical and scanning electron microscopy, a sequence of structural changes is observed after incubation with cystic fibrosis serum; this sequence does not occur with normal serum. The earliest changes involve swelling of the goblet cells, with subsequent discharge of mucus onto the epithelial surface. This is followed by disruption of the normally rapid and synchronized ciliary activity. Mucus gradually extends over the surface entangling cilia. Finally, some shedding of ciliated cells occurs from the epithelium. These findings suggest that factors in cystic fibrosis serum cause discharge of mucus leading to a disturbance of the normal ciliary activity in the rabbit lung. It is postulated that such changes result in dysfunction of the mucociliary clearance mechanism and that this dysfunction may be a contributory factor to the pathogenesis of lung disease.