Tritiated thymidine autoradiographic study on the origin and renewal of argentaffin cells in the pyloric gland of hamsters

Summary The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with ³H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt.By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with ³H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus.The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the random loss system. The half time of turnover of this cell population was 15 days on average.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

Summary The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. Transitional cells, presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Tritiated thymidine autoradiographic study of cell migration and renewal in the pyloric mucosa of golden hamsters

Summary The kinetics of cell proliferation, migration and renewal in the pyloric mucosa of golden hamsters were studied by flash, cumulative and pulse labelling autoradiography following ³H-thymidine injections.By flash labelling autoradiography, it was shown that the labelled epithelial cells are exclusively confined to a zone several cells wide in the region of the isthmus between the gastric pits and the pyloric glands. In the cumulative and pulse labelling experiments, this cell proliferation in the isthmus region was shown to be for replacement of both the surface epithelial and the glandular cells. The surface epithelial cells of the pyloric mucosa arising in the upper portion of the isthmus come to line the pits and the surface, and are sloughed off into the gastric lumen within a week. The mucin-containing glandular cells, which arise more deeply in the isthmus region, migrate downwards and are apparently lost at the deepest level of the glands. The life span of the mucin-containing glandular cells was estimated at about 14 days. This cell type appears to undergo renewal of the first produced, first lost pipe line variety. However, a small number of glandular cells was found to survive for more than 20 days (up to 30 days), suggesting the existence of a sub-population of cells with different kinetics in the pyloric glands.Supported by a Grant-in-Aid for Cancer Research from Ministry of Education, Science and Culture, Japan     

Cell population kinetics of zymogen and parietal cells in the stomach of mice

Summary With autoradiography after labelling with tritiated thymidine, the kinetics of zymogen and parietal cells were studied in the gastric mucosa of mice. After one intraperitoneal injection of the DNA precursor, zymogen cells in the DNA synthesis phase were clearly identified on autoradiograms, whereas no parietal cells were seen to synthesize DNA.In another group of mice, multiple injections were used in order to obtain a greater number of labelled cells. Following the latter procedure, analysis of grain count distributions over labelled zymogen cells and of labelling indices allowed detection of two subsequent zymogen cell divisions within an interval of approximately two months. This indicates that the cell turnover of zymogen cells is at least partly assured by their own mitotic activity.By contrast, parietal cells showed no evidence of cell division, but appeared to be derived through differentiation from other cells in the neck area of the gland. Analysis of spatial distribution of the labelled parietal cells in the glandular tube indicated that, in time, most newly formed parietal cells undergo a slow migration directed downwards to the bottom of the fundic glands.These results clearly show that the zymogen and the parietal cell population of the fundic glands have a different kinetic behaviour.This work was supported by a grant of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog,Rana temporaria,with special reference to physiological regeneration of the preoptic nucleus

The localization and fine structure of proliferating cells in the hypothalamic preoptic area were studied by light-and electron-microscopic radioautography 1–2 h following single application of ³H-thymidine to adult Rana temporaria taken from their natural habitat in the spring and autumn. ³H-thymidine uptake by proliferating cells was much more pronounced in frogs caught in May/June, i.e., a month after the breeding period (labeled cells represent about 10% of the total ventricular zone cell population), compared to animals caught in mid-September, when it was very low. In both ³H-thymidine treatment groups the vast majority of labeled cells are found exclusively within the preoptic recess ventricular zone. With regard to ultrastructure, it contained proliferating cells of at least 4 types, ranging from immature forms (bipolar stem cells) to more differentiated elements (tanycyte-like ependymoblasts, classical ependymoblasts). All of them showed label over their nuclei indicating that these cells are capable of DNA synthesis and mitosis. The possible role of the preoptic recess ventricular zone as a source of precursor cells for new peptidergic neurosecretory cells, conventional neurons and glial cells in the hypothalamic preoptic area of the adult frog is discussed.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype

The presence and time-course of -adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [¹²⁵I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the ₁-subtype of the receptor, whereas the ₂-subtype binding remained constant up to 3 weeks in vitro. The delay of ₁-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased -adrenoceptor levels could be induced by chronic treatment of the -agonist isoproterenol (1 M) introduced either for 3 or 13 days. Again, changes in density were found only for the ₁-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.Special issue dedicated to Dr. Robert Balázs     

Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D<Subscript>3</Subscript>

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34⁺ progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34⁺ cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D₃ could diminish CD34⁺ cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 g) of 25-hydroxyvitamin D₃, with an emphasis on the six patients who received the maximum dosage of 60 g per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D₃ reduces the number of immune suppressive CD34⁺ cells, increases HLA-DR expression, increases plasma IL-12 and IFN- levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D₃ treatment did not modulate plasma IL-1, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF- levels.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - high CD34⁺ patients patients with greater than 1% baseline CD34⁺ cell levels - HLA human leukocyte antigen - IFN interferon - IL interleukin - low CD34⁺ patients patients with less than 1% baseline CD34⁺ cell levels - OD optical density - TGF transforming growth factor     

Cadmium‐induced apoptosis and phenotypic changes in mouse thymocytes

At present cadmium (Cd)induced immunotoxicity and the mechanisms involved have not been fully elucidated. The main objective of the present study is to explore the apoptogenic property of Cd in primary cultured mouse thymocytes and its effect on cell surface marker expression and phenotypic changes. Cdinduced thymocyte apoptosis was determined by TdTmediated dUTP nick end labeling (TUNEL) assay, DNA content/cell cycle analysis and DNA gel electrophoresis. The results showed that Cd was able to cause apoptosis in mouse thymocytes in a time and dosedependent manner. Moreover, different subsets of thymocytes possessed different susceptibility to the apoptotic effect of Cd, in the order of CD8⁺ > CD4^–CD8^– (double negative cells, DN) > CD4⁺CD8⁺ (double positive cells, DP) > CD4⁺. Cd treatment also altered thymocyte surface marker expression, leading to evident phenotypic changes. Such changes were characterized by a decline in DP cells and a marked decrease in CD4⁺/CD8⁺ ratio, mainly due to a significant increase in CD8⁺ subsets. These observations help to obtain a better understanding of the immunotoxic and immunomodulatory effects of Cd.     

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Immunocytochemical study of pepsinogen 1-producing cells in the fundic mucosa of the stomach in developing mice

Summary Development and maturation of pepsinogen 1-producing cells were studied in the gastric fundic mucosa of the mouse by means of light- and electron-microscopic immunocytochemistry using rabbit anti-rat pepsinogen 1-serum. In the adult mouse, secretory granules in mucous neck cells, transitional mucous neck/chief cells and chief cells are immunolabeled. The numerical density of gold particles on zymogen granules is not significantly altered among different stages of maturation of chief cells. In addition, rough endoplasmic reticulum and Golgi complex of these cell types show a weak labeling. In mice from day 16 of gestation to postnatal day 14 mucous neck cells and chief cells cannot be distinguished, but only one type of pepsinogen 1-producing cell, called primitive chief cell, is identified in the fundic gland. The intensity of immunoreactivity of secretory granules in primitive chief cells is uniform within an individual cell but varies greatly among different cells. The majority of primitive chief cells contains weakly labeled granules regardless of the maturation stage of cells or of animals. On postnatal day 21, mucous neck, transitional and chief cells are distinguishable, and secretory granules in these cells are intensely immunolabeled as in the adult. These results suggest that pepsinogen 1-production rapidly increases with differentiation of mucouse neck and chief cells.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) by organogenesis

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 M) in combination with naphthaleneacetic acid(0.05 M) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 M) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 M), indole-3-butyric acid (1.2 M) and benzyladenine (0.4 M). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - GD Gresshoff and Doy (1972) nutrient media - IBA Indole-3-butyric acid - KN Kinetin - NAA -Naphthaleneacetic acid - 2iP 2-isopentenyl adenine - ZN Zeatin     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na⁺,K⁺-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na⁺,K⁺-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220–250 m, and in the labyrinth, at EI 350 m. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410–440 m), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na⁺,K⁺-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.We thank the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran for financial aid and support. Special thanks are also due to the Société Française dExportation des Ressources Educatives (SFERE) for the scholarship to S.K.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves

We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3--glucosidase (-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 l·l^-1, 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic -1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and -1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.Abbreviations IgG immunoglobulin G - IWF intercellular wash fluid - PBS 0.14 M NaCl, 0.1 M K₂HPO₄, pH 7.5 - TMV tobacco mosaic virus We thank Monique Seldran and Alfred Milani for expert technical help, Patricia Ahl-Goy, Ciba-Geigy, AG, Basel for supplying IWF from TMV-infected leaves, and our colleagues Thomas Boller and Lilian Sticher for their comments and criticism.