Morphological and cytochemical modifications induced by colchicine treatment in the duodenal epithelium of mouse

Summary S.W.R. adult male mice received one injection of colchicine and were killed at various time-intervals from 4 to 24 hours. Ultrastructural morphology and cytochemical activity of acid phosphatase, arylsulfatase and thiamine pyrophosphatase were recorded in the duodenal epithelium. Extensive damage was observed in the undifferentiated cells with formation of large heterophagosomes and in the absorptive cells with apparition of cytolysomes. The activity of the acid phosphatase and thiamine pyrophosphatase decreased after 6 hours of treatment. Moreover, the migration of the acid phosphatase content of the Golgi vesicles was restraint. These observations were related to the disruptive action of the alcaloid towards the microtubules.This work was supported by a grant from the Medical Research Council of Canada.Mr. Raymackers was a recipient of a studentship of the Medical Research Council of Canada. The authors are greatly indebted to Mr. Michel Couture and Pierre Magny for their skilful technical aid.     

Electron microscopic histochemistry of lysosomes in neurosecretory nerve endings and pituicytes of rat posterior pituitary

Summary Electron microscopic localization of acid phosphatase (AcPase) was carried out on posterior pituitary glands from rats. An estimated 5% of the neurosecretory nerve terminals contained structures which showed reaction product. Most of the lysosomes were small dense bodies, often with a membranous substructure. Other lysosomes were larger in size or were found within vacuoles. AcPase was also localized to lysosomes and the Golgi apparatus of pituicytes. Evidence is presented which would associate the large lipid droplets characteristic of pituicytes with AcPase-positive dense bodies. The present study indicates that hydrolytic activity by lysosomes occurs within the terminals of neurosecretory cells, and adds further support to the concept that this process represents a normal phenomenon of cells and their extensions in general.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developingRana Catesbeiana

Summary Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog,Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.This work was supported by grants from the France-Quebec agreements (J. Hourdry) and from the Medical Research Council of Canada (J.S. Hugon)     

Mouse intestinal sensitivity to cycloheximide

Summary Cycloheximide 40 mg/kg was injected into adult male mice. Biochemical, histochemical and morphological modifications of the mucosal cells of the duodenum have been recorded after one or several injections. Biochemical experiments have shown a rapid decrease of alkaline phosphatase activity in the duodenum and a significant increase in the liver. Histochemical studies have shown a total disappearance of the alkaline phosphatase activity in the Golgi zone of the absorbing cells, two hours after one injection. Acid phosphatase, thiamine-pyrophosphatase and glucose-6-phosphatase did not exhibit the same pattern. Alterations of the ultrastructure of the villus cells have been observed after several injections. Atrophy of the Golgi cisternae, appearance of autophagic vacuoles and decrease of the goblet cells secretory granules have been the most frequent modifications.This work was supported by grants from the Medical Research Council of Canada and from the National Defense Board of Canada.The authors are greatly indebted to Mr. M. Couture for his skillful technical help.Mr. Charuel is a recipient of a studentship from the France-Quebec agreement.     

Kinetic study of serotonin metabolism in the suprachiasmatic nucleus of the rat: neuroendocrine incidence (author's transl)

The suprachiasmatic nucleus (SCN), which is implicated in the regulation of several cyclic neuroendocrine rhythms, displays a conspicuous and apparently specific serotoninergic innervation. Our study was intended to establish more precise correlations between the incidence of serotonin (5 HT) metabolism in the activity of the SCN, and neuroendocrine rhythms. For this purpose, castrated female rats, having subcutaneous implants of estradiol, were studied. These animals display very marked circadian fluctuations in plasma levels of the pituitary hormones ACTH, LH and PRL; a relatively well-synchronized increment in all these hormones occurs between 11.00 and 15.00. Punches were obtained to determine the endogenous content of 5 HT, measured by a radioenzymatic technique, simultaneously in SCN and median eminence (ME). An index of SCN activity was determined from in vivo SCN 2-deoxy (1-14C) glucose (DG) uptake; the retention was estimated on radioautographs. Endogenous level of 5 HT displayed a marked circadian rhythm with a peak between 12.00 and 15.00 in the SCN; 5 HT levels were constant throughout the day in the ME. 14C-DG uptake was greater at 15.00 than at 9.00. However, after PCPA treatment or raphe lesions, the uptake of 14C-DG was the same at 9.00 and 15.00. Taken together, these results strongly suggest that in our model: (1) SCN displays a rhythm of activity in the light period; (2) SCN displays specific rhythms in the content of 5 HT; (3) the SCN rhythm of activity must be under an inhibitory control of 5 HT, since the lowest metabolic level is increased at 9.00 by inactivation of 5 HT system; (4) the close relationships between the initial release phase of several pituitary hormones, the increase of metabolic activity in the SCN and the higher level of the 5 HT in the nucleus suggest that 5 HT terminals in the SCN play an important part in the control of cyclic hormonal secretion.     

The effect of variable mechanical impedance on the control of antagonistic muscles

Most models of muscle have assumed that the series elastic element has a constant stiffness, although experimentally this is not true, either at the level of individual cross-bridges or that of the whole muscle. In this paper elastic and viscous elements are introduced whose properties vary systematically with activity levels as has been found experimentally. The effects of these nonlinear variations on the methods for optimally controlling a pair of antagonistic muscles are calculated. Although the nonlinearities add some complexity to the derivation and are important to the mechanism of force generation in the muscle, they do not qualitatively change the overall control of movement.This work was partly supported by the Natural Sciences and Engineering Research Council of Canada NSERC OGP-4345 and by the Medical Research Council of Canada Grant PG-47 through the University of Alberta     

Pyruvate kinase binding to particles in brain

Interaction of pyruvate kinase with membrane fractions of rat brain homogenates was shown to be electrostatic. Ionic compounds readily solubilized the enzyme from membranes, and the enzyme was more readily solubilized at high pH versus low pH. Developmental patterns indicated that the particulate and soluble forms of pyruvate kinase increased nearly in synchrony from birth until maturity.The author is grateful for the technical assistance provided by Dolores Suga. This research was supported by a grant (MA 5447) from the Medical Research Council of Canada.     

Smooth endoplasmic reticulum proliferation in mouse enterocytes induced by fructose feeding

Summary Adult male mice receiving a fructose diet show, after three days, a large proliferation of the smooth endoplasmic reticulum in the jejunal absorptive cells. This extension is a secondary stage of the trememdous increase of the glucose-6-phosphatase activity and seems to result from a crypt regulation. Glucose feeding does not provoke the same morphologic changes. This work was supported by a research grant from the Medical Research Council of Canada. Dr. D. Maestracci Ph. D. is a recipient of a Centennial Fellowship of the Medical Research Council of Canada. Mr. D. Ménard is a recipient of a studentship of the Medical Reserach Council of Canada. The authors are greatly indebted to Mrs. S. Sinave for her skillful technical assistance.     

Ultrastructural localization of acid phosphatase in the posterior pituitary of the dehydrated rat

Summary Acid phosphatase (AcPase) was localized by means of electron-microscopic histochemistry and estimated biochemically in the posterior pituitary of rats deprived of water, given 2% NaCl ad libitum, or given tap water ad libitum over 6 days. Autophagic vacuoles, some of which gave a positive AcPase reaction, often contained neurosecretory granules (NSG) in nerve endings of control animals on tap water. Nerve endings of water-deprived or salt-treated rats were depleted of NSG, but frequently contained dense membranous residual bodies, some of which appeared to enclose microvesicles. Smooth endoplasmic reticulum located in axons and terminals appears to be a source of hydrolytic enzymes for neurohypophysial lysosomes. The total amount of AcPase per posterior lobe increased progressively to 40% above control levels after 6 days of water deprivation or salt administration, and this increase may reflect accelerated production of neuronal components in neurohypophysial cells whose secretory rate has been stimulated by elevated body osmolarity.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Theoretical and experimental derivation of the function relating the size distribution of spherical particles in a volume element to that obtained from images of sections taken through it

Summary The mathematical formulation of three classes of theories concerning the problem outlined in the title of this study was critically assessed and experimentally evaluated. It was found that only one class of the theories describes the relation between the distribution of particles in a volume element to that observed in sections through this volume in a satisfactory way. The errors in the remaining two classes could be traced and the corrections made after a mathematically rigorous analysis brought these theories to comply with a model which, as we could prove experimentally, deserves a high level of confidence.Supported by a grant from the Medical Research Council of Canada.In partial fulfillment of the requirements for obtention of the degree of a Master of Science. Supported by a Studentship from the Medical Research Council of Canada.Scholar of the Medical Research Council of Canada.     

Red-light-enhanced conversion of tritiated gibberellin A9 into other gibberellin-like substances in homogenates of etiolated barley leaves

Summary Irradiation of homogenates of etiolated barley leaves with red light resulted in an increase in the levels of gibberellin (GA)-like substances as compared to dark controls. When homogenates were fed with [³H]-GA₉ there was as incorporation of the radioactivity into a number of other GA's: this process occurred to a greater extent in red light than in darkness, and could be inhibited by boiling the extract prior to addition of the [³H]-GA₉.Supported by National Research Council (Canada) grant A-5727 (Dr. D. M. Reid).Supported by National Research Council (Canada) grant A-2585 (Dr. R. P. Pharis).Supported by NATO-Science Research Council (U. K.) Postdoctoral Fellowship.     

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla

Summary The synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-³H and L-3,4-dihydroxy [ring 2,5,6-³H] phenylalanine (L-dopa³H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine ³H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine ³H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa ³H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine ³H or L-dopa ³H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals. Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group of Hypertension of the Clinical Research Institute of Montreal and by the Canadian Heart Foundation     

Organ culture of adult mouse intestine

Summary Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase is measured during the first 24h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.This work was supported by research grants from the Medical Research Council of Canada (J.S.H.) (D.M.)Mr. Chabot is a recipient of a studentship from the Medical Research Council of CanadaD. Ménard, Ph.D. is chercheur-boursier from the CRSQ     

Staining properties of duodenal mast cells in 48/80-treated rats

Summary Mast cells in the tongue, mesentery and lamina propria of the duodenal mucosa in normal and 48/80-treated rats were observed at different time intervals. The tissues were studied comparatively after staining with toluidine blue, acridine orange or alcian bluesafranin. Under the experimental conditions used, the mast cells in the tongue and mesentery showed constant positive reactions to toluidine blue and acridine orange, both of which failed to demonstrate the presence of mast cells in the lamina propria of the duodenal mucosa. The combined alcian blue-safranin stain elicited a safranin-positive reaction in the mast cells of the tongue and mesentery and an alcian blue reaction in those of the lamina propria of the duodenal mucosa. This alcianophilia of the duodenal mast cells was not affected by compound 48/80. On the other hand, the safranin stain of the tongue and mesentery mast cells was altered to alcian blue by the drug. The results are discussed in the light of recent developments in mast cell research.This work was supported by grant MA-2236 of the Medical Research Council of Canada.     

Genetic characterization of methotrexate-resistant chinese hamster ovary cells

Summary In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 × 10⁻⁹ mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Classes I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This research was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada and the National Institutes of Health of the United States. W. F. was a Postdoctoral Fellow of the Medical Research Council of Canada.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

Cytomembranes in first cleavage xenopus embryos

Summary The ultrastructure and interrelationships of the Golgi body, endoplasmic reticulum and lipid droplets have been studied in the first cleavage Xenopus embryos. Lipid droplets, usually spherical or sometimes multilobed, did not have a discernible limiting membrane, although some had an incomplete electron dense partition. The Golgi bodies and endoplasmic reticulum were seen continuous with lipid droplets and the profiles indicated a probable formation of these membranes from lipid droplet material. Rough endoplasmic reticulum (ER) mainly consisted of paired tubular cisternae and vesicles containing filamentous material that gave a fringed appearance. The relationships of paired cisternae with the Golgi body suggested a transformation of ER membranes into the Golgi body membranes. In addition, paired ER cisternae showed a close apposition with the limiting membrane of the yolk platelet. Lone ER cisternae that contained moderately electron dense material instead of filaments were also present and showed numerous associated vesicles near the Golgi body. The Golgi body showed several morphological forms including a single fenestrated cisterna, two to four flat or cup-shaped cisternae, or up to seven cisternae, some of which were dilated and similar to fringed ER in appearance. These forms could be different developmental stages of the organelle. Coated vesicles were seen continuous with the cisternae of the Golgi body. A probable route for the assembly of the cell surface material has been proposed.This work was supported by a grant from the Medical Research Council of Canada to one of us (E.J.S.).     

Purification and properties of two distinct groups of ADH isozymes from Chinese hamster liver

Two major classes of alcohol dehydrogenase isozymes have been purified from Chinese hamster liver. The two isozyme classes have the same subunit molecular weights but different electrophoretic mobilities. They have a similar range of substrates but different KROM values and sensitivities to the inhibitor pyrazole. The ADHs are immunologically related as determined by Ouchterlony double diffusion experiments. These results suggest that the isozymes are encoded by different structural gene loci derived from a common ancestral gene.Financial support was provided by the National Cancer Institute of Canada and the Medical Research Council of Canada, Grant MT4860. J.-P. Thirion is a Research Scholar of the Science Research Council of Quebec.     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

Percoll centrifugation eliminates mold contaminants from cell cultures

This work was supported by a grant and research associateship to N.A. from the Medical Research Council of Canada and the National Cancer Institute of Canada.