Ultrastructural observations on some membranous cytoplasmic inclusion bodies in follicular cells of experimentally-induced goitres in tadpoles and toads of Xenopus laevis Daudin

Summary Tadpoles and young toads of Xenopus laevis were maintained in aqueous solutions of potassium perchlorate or thiourea (0.005% or 0.01% w/v) for up to 20 months. Metamorphosis of tadpoles was inhibited. Within a short time large well-vascularized goitres developed in both tadpoles and toads. An ultrastructural investigation of some of the follicular epithelial cells of these goitres revealed some unusual cytoplasmic membranous inclusions and a morphological account of these structures is presented. It is suggested that some of these complex membranous inclusions may give rise to cytosomes. The relationship between these complex cytoplasmic organelles is considered together with their possible significance and role in thyroid cell functioning.This work was carried out during the tenure by one of us (R.C.) of a Medical Research Council Scholarship and forms part of a programme of research in amphibian thyroid physiology supported by the Medical Research Council to whom we are indebted for their generous assistance.     

Ultrastructural observations on the droplets of experimentally-induced goitres in Xenopus laevis with especial reference to the development of uhlenhuth colloid cells

Summary Goitres were induced in tadpoles and young toads of Xenopus laevis Daudin by prolonged immersion in aqueous solutions (0.005% or 0.01% w/v) of potassium perchlorate, potassium thiocyanate, thiourea or propyl-thiouracil. Various categories of single membrane-limited droplets were induced and these are described. Also some of the goitrous cells showed an accumulation of colloid droplets leading to the formation of Uhlenhuth colloid cells. The development and significance of these cells in the amphibian goitres is considered.This work was carried out during the tenure by one of us (R. C.) of a Medical Research Council Scholarship and forms part of a programme of research in amphibian thyroid physiology supported by the Medical Research Council to whom we are indebted for their generous 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.     

Cellular defense in an Arthropod in response to infection with aSalmonella typhimurium strain

Larvae ofGalleria mellonella L. when injected with cells ofSalmonella typhimurium strain LT 2 responded by cellular defense: their hemocytes gathered and formed a pseudo-tissue by which the bacteria were encapsulated. In contrast,S. typhimurium strain 7 Suc LL caused lysis of about 64% of the hemocytes and cellular defense against this strain was lacking.This explains the difference in the mortality rate of the larvae which was 10% after intracoelomic injection with 2×10⁴ cells of strain LT 2 and 48% with the same number of cells of strain 7 Suc LL.After injection of strain 7 Suc LL, lysis of hemocytes preceded proliferation of bacteria; moreover such lysis also occurred after injection of cell-free culture filtrate of this strain. This suggests that lysis is due to either a toxic substance or a proteolytic enzyme produced by strain 7 Suc LL.These investigations were supported by the Medical Research Council of Canada Grant MA-2385 and the National Research Council of Canada Grant A-3746.     

Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults

Summary Improved methods for rearing and screening large numbers of flies permitted the recovery of 10 mutations exhibiting a reversible temperature-dependent adult paralysis among 1.1×10⁶ flies tested. Of the 10 mutations, two were allelic to para ^ts,two were alleles in a new locus, stoned (stn), and six fell into a third area, the shibire (shi) locus. Several of the shi alleles cause embryonic, larval and adult paralysis at 29° C as well as structural anomalies of various tissues. In addition to the ts mutations, several non-conditional mutations affecting adult movement were recovered.This work was supported by the National Research Council of Canada, grant A-1764, National Cancer Institute of Canada, grant 6051.Medical Research Council of Canada Postdoctoral Fellow.     

Proteins of the Drosophila melanogaster male reproductive system: Two-dimensional gel patterns of proteins synthesized in the XO,XY, and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins

Testes and paragonial glands of Drosophila melanogaster wild-type males were labeled in vitro using [³⁵S]methionine, and the proteins synthesized were analyzed by 2-dimensional gel electrophoresis. Testes and paragonial glands were also labeled in vivo by feeding male larvae ³⁵S-labeled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. [³H]Proline was also used to label testes, and no significant differences from the ³⁵S pattern were noted. Testes and paragonial glands from XO and XYY males were labeled in vitro with [³⁵S]methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Pure sperm were dissected manually from in vivo labeled males and the proteins analyzed. Ninety-two proteins were detected, which were all synthesized in comparable amounts by XO, XY, and XYY males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational or regulatory proteins present only in very small amounts in the adult testes. ³⁵S-labeled males were also mated to unlabeled females and the transferred proteins analyzed on two-dimensional PAGE. The contributions of the testis and paragonial gland to the ejaculate were determined.This work was supported by a grant from the Medical Research Council of Canada and by the U.S. National Institutes of Health (Grant No. GM-22753). J. I. B. is the recipient of a Medical Research Council of Canada studentship.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

The stabilisation of a transient mutagen-sensitive state in Neurospora by the protein synthesis inhibitor actidione

Summary Experimental evidence has been obtained to show that a transient mutagen sensitive state, believed to be induced in Neurospora by DEB, can be stabilised by the protein synthesis inhibitor actidione. Sensitisation can thus be separated from the complicating effects of traces of the DEB retained by the cells following washing. The bearing of these results on the interpretation of the DEB after-effect and DEB mutation induction curves is briefly discussed.Research supported by the Medical Research Council.     

DNA amounts in the nuclei ofParamecium aurelia andTetrahymena pyriformis

DNA amounts have been determined in the micronuclei and macronuclei of 8 strains ofParamecium aurelia and 6 strains ofTetrahymena pyriformis. In the case ofTetrahymena a distribution of values for the amount of DNA in the macronuclei of all the strains was observed but the lowest values were approximately the same, viz. 1.17×10⁻¹¹ g. There are two groups of strains in relation to micronuclear DNA values ofTetrahymena, one giving an average of 0.36×10⁻¹² g and the other 0.815×10⁻¹² g. The ratio of MIC/MAC DNA varies in the two groups.Paramecium again has a range of macronuclear values within each stock—lowest value 2.51×10⁻¹⁰ g—and the micronuclear values are similar in all stocks—approximately 0.613×10⁻¹² g. The ratio of MIC/MAC DNA is similar in each stock.—The haploid genome values calculated from these data show excellent agreement with the values obtained by DNA renaturation studies. Supported by a Research Grant B/SR/8276 from the Science Research Council. The Vickers densitometer was purchased with a grant from the Medical Research Council.     

Use of immunocytochemical staining of somatostatin for correlative light and electron microscopic investigation of D cells in the pancreatic islet of Xiphophorus helleri H. (Teleostei)

Summary Somatostatin-containing cells have been demonstrated by immunocytochemistry in semithin sections of the pancreatic islet of the teleost fish, Xiphophorus helleri. These cells were shown by correlative light and electron microscopy to be identical with D cells previously defined in this species by the silver impregnation method of Hellman and Hellerström.Supported in part by grants from the British Council and from the Medical Research Council of Great Britain     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

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)     

The accumulation of lactic acid and its influence on the growth ofPlasmodium falciparum in synchronized cultures

Summary Synchronization ofPlasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66±0.55 μmol LA per 12 h per 10⁷ parasitized cells (n=26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72±0.62 μmol LA per 12 hours per 10⁹ red blood cells) (n=41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth ofPlasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield. J. W. Z. was supported during part of this study by a long-term fellowship of the European Molecular Biology Organization, Heidelberg, West Germany, followed by a Research Associateship from the National Research Council, Washington, D.C. The project was supported by grants from the Medical Research Council to J. G. S. and by the Naval Research and Development Command, Work Unit No. 3M 162 770 A871 AE 312. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large.     

Comparative studies on human skin fibroblasts: Life span and lipid metabolism in medium containing fetal bovine or human serum

Summary Three strains of human skin fibroblasts were cultivated in nutrient medium supplemented either with human serum or fetal bovine serum, and growth and lipid synthesis were compared. Rates of cellular growth were similar in both kinds of medium, but the replicative life spans of all three strains were curtailed significantly in human-serum medium. Incorporation of label into the major classes of neutral lipids from [¹⁴C]acetate and³H₂O was increased also in human-serum medium. Since human serum contained higher concentrations of cholesterol known to reduce endogenous cholesterol synthesis, these results were unexpected. Nonlipid factors in human serum may account for the shortened cellular life spans and increased lipogenesis and perhaps for the potential to develop atherosclerosis. Supported by grants from the Ontario Heart Foundation and Medical Research Council of Canada during the tenure of a Senior Research Fellowship from the Ontario Heart Foundation (J.T.C.) and a Scholarship from the Medical Research Council of Canada (S.G.).     

An allele (Pk-1 b ) from wild-caught mice that affects the activity and kinetics of erythrocyte and liver pyruvate kinase

A true breeding strain was made from a wild-caught mouse with low erythrocyte pyruvate kinase (E.C. 2.7.1.40) activity. This variation showed additive inheritance and segregated as an allele at a single locus (Pk-1 ^b). Mice homozygous for the reduced blood pyruvate kinase activity cosegregated for reduced liver activity. In both these tissues the variant enzyme had a lowered heat stability and reduced K _m values for ADP. An increased stimulation by FDP was also detected in the liver pyruvate kinase. No difference in the isoelectric point of the variant enzyme in either erythrocyte or liver was observed when compared with the enzyme from C57BL mice (Pk-1 ^a/Pk-1 ^a). It is concluded that Pk-1 is the structural gene for the erythrocyte and the major liver pyruvate kinase. No other tissue pyruvate kinase showed altered characteristics.This work was supported by a Medical Research Council grant.     

Stable acid phosphatase: I. Demonstration and distribution

Summary After a pH-dependent reactivation a highly stable form of acid phosphatase (SAPhase) could be demonstrated in active cells of the macrophage/giant cell/osteoclast series and also in epiphyseal chondrocytes, in cells lining bone undergoing resorption and in hamster cosinophils. Because acid phosphatases of epithelial cells in rat, hamster and Macaca sp. tissues did not possess this stability, SAPhase served as a useful cell marker for the above mesenchymal cell types in paraffin and glycol-methacrylate sections even after rapid demineralization in acidic buffers. Conformational alterations appear to occur in the enzyme during formaldehyde fixation, embedding, and reactivation. The granular staining of SAPhase and the successful use of a non-aqueous fixative suggest an association of SAPhase with lysosomes and their membranes. Cells of mesenchymal origin that are actively engaged in intra- and/or extracellular digestion contain high levels of SAPhase. The distribution and properties of SAPhase indicate an interrelationship between mononuclear and mutinuclear cell types actively engaged in such digestive processes.Supported by the Medical Research Council of Great Britain     

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     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.