Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Structure of isolated sensilla and sensory neurons in vitro: Observations on developing silkmoth antennae

Summary By combined enzymatic and mechanical treatment, it was possible to dissociate the sensory epithelium of developing antennae of male Antheraea polyphemus and A. pernyi silkmoths from the stage of separation of the antennal branches up to the early stages of cuticle deposition. Large numbers of entire developing trichoid sensilla were isolated. These are characterized by a large trichogen cell with a long apical, hair-forming process and a large nucleus. A cluster of 2–3 sensory neurons, enclosed by the thecogen cell, is situated in the basal region. The dendrites run past the nucleus of the trichogen cell into the apical process from which they protrude laterally. The nuclei of the tormogen and a 4^th enveloping cell can be distinguished near the base of the prospective hair. After further dissociation, only the neuron clusters remain, still enclosed by their thecogen cell and often attached to the antennal branch nerve via their axons. It is finally possible to disrupt the thecogen cells and the axons, leaving the sensory neurons with inner dendritic segments and axon stumps. The majority of these neurons can be expected to be olfactory.     

Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.     

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.Abbreviations BSA bovine serum albumin - Con A concanavalin A - SDS sodium dodecyl sulphate     

Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

Summary A novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc ⁺ cells; conversely the division rate of cfcA1 cells was higher than that of cfc ⁺ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.     

Reduction in the free indole-3-acetic acid levels in Alaska pea toy the gibberellin biosynthesis inhibitor uniconazol

The gibberellin biosynthesis inhibitor uniconazol reduces both the elongation and indole-3-acetic acid content of growing Pisum sativum cv. Alaska intemodes. Both internode growth and indole-3-acetic acid content in uniconazol-treated plants can be elevated by gibberellin A3 treatment. The lengths of the growing intemodes are directly related to the indole-3-acetic acid contents.     

Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Local and systemic changes in gene expression induced in tomato plants by wounding and by elicitor treatment

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants have been wounded to induce the accumulation of proteinase-inhibitor proteins (PI proteins) at the local site of injury and systemically in unwounded tissues. To determine the range of genes affected in the wound-response, polysomal mRNA has been isolated from the damaged leaves and from systemically responding leaves over a time-course of 2, 4, 10 and 24 h after wounding. Changes in the pattern of ³⁵S-translation products indicate that the events that occur at the local wound-site are different from those that occur systemically, both with respect to the number of genes that are regulated and the timing of their regulation. In order to compare the effects of wounding and an endogenous systemic signal generated at the wound-site with those of elicitor (proteinase-inhibitor-inducing factor, PIIF) treatment of excised plants, polysomal mRNA has also been isolated from leaves of plants over a time-course of 2, 4, 10 and 24 h after PIIF-treatment. Changes in the pattern of ³⁵S-translation products indicates that the events induced by PIIF resemble those induced by mechanical injury, rather than those induced by the endogenous systemic signal.Abbreviations IFF isoelectric focussing - PI proteins proteinase inhibitor proteins - PIIF proteinase-inhibitor-inducing factor - ssRubisco small subunit of ribulose-1,5-bisphosphate carboxylase