Significance of bacterial ectoenzymes in aquatic environments

The report presents studies on temporal and spatial variations of kinetics (Vmax and Km) of bacterial ectoenzyme activity (-glucosidase - Glc, leucine aminopeptidase - Leu-amp) in the naturally eutrophic Plusee. Glc and Leu-amp activity were positively correlated with the flux of polymeric materials (polysaccharides, proteins) in the lake. Glc activity was low when algal populations grew actively, but during the algal bloom breakdown Glc activity increased rapidly. Leu-amp displayed the highest rates of activity in the epilimnion and was tightly coupled to bacterial production. The synthesis of studied ectoenzymes was under control of a repression/derepression mechanism. The significance of ectoenzymes for the transformation and bacterial utilization of organic matter, and their role in the microbial loop in aquatic environments is discussed.     

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture

Post-embedding immunocytochemical techniques using peroxidase–antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9--D-ribofuranosil-N ⁶-(²-isopentenil) adenina ([9R]iP), 9--D-ribofuranosyl-zeatin ([9R]Z) and 9--D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72h in liquid medium. Subcellular immuno-localization was performed in cells from explants grown for 35d on agar solidified-medium and for 30min, 4 and 16h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4h of culture in the cytoplasm, and at 16h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.     

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, dimethyl--cyclodextrin and hydroxypropyl--cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium.The complexed lignan disappeared rapidly from the culture medium, within 24h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin--d-glucoside. A maximal bioconversion rate of 0.51 mmol l^-1 suspension day^-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl--cyclodextrin complex at a final concentration of 1.35 mM.     

The multiplication constant of a microorganism in a colony is normally reduced by irradiation but still remains as a characteristic constant-A new approach to determining irradiation pasteurization doses

This work is based on a previous observation and on a related mathematical modeling regarding the linear growth of a colony of microorganisms under given conditions. We had previously shown that the growth rate of the colony is merely proportional to the individual exponential multiplication constant, , of the microorganisms.Tiny colonies of penicillium are subjected to different doses of irradiation. The subsequent observation of the colonies' growth rate beautifully furnishes a measure of how the multiplication constant, , of the microorganism is affected by irradiation.The plot of with respect to the irradiation dose, shows a linear interdependence between the two quantities. The extrapolation of this plot easily yields the radiation pasteurization dose of the microorganisms in hand.     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Vorläufige Versuche über die Elastizität des Atraktoplasmas

Summary Spindles of nuclei mostly from metaphases of expiants in vitro, Set free according to Dan's method by means of solutions of Duponol (a sodium lauryl sulfonate), have been studied according to their elasticity. The isolated atractoplasm has shown a static modul of elasticityE0.8 to 1.6×10⁶ dynes×cm^–2, and, sometimes, a Poisson number of0.42, that is to say the atractoplasmic gel of the spindles of nuclei is certain elastically. The values of the modul of elongation agree satisfactory with those values recently ascertained by Crick and Hughes with cytoplasm oi explanted cells.     

Cytochemical localization of nicotinamide adenine dinucleotide phosphatase (NADPase) in bovine Leydig cells

Summary The ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) in bovine Leydig cells has been studied and compared with the pattern of thiamine pyrophosphatase (TPPase) and acid phosphatase distribution in these cells. Using -nicotinamide adenine dinucleotide phosphate (-NADP⁺) as substrate, a marked staining is observed in the intermediate Golgi saccules with some focal extension to the trans aspect. Cisternae on the cis side and associated vesicles yielded only slightly positive reactions. The pattern of NADPase localization is clearly different from that of TPPase which consistently stains only the trans Golgi elements. The specifity of NADPase for its substrate, -NADP⁺, was clearly demonstrated by using substrates modified in either the nicotinamide region e.g. -nicotinamide adenine dinucleotide phosphate (-NADP⁺), -thionicotinamide adenine dinuclcotide phosphate (Thio-NADP⁺), in the attachment site of the monoester phosphate group to the molecule (e.g. 2 monophospho-adenosine 5-diphosphoribose (ATP-ribose) or adenosine-5-monophosphate (5AMP). With these substrates only weak or negative reactions were obtained in the Golgi apparatus of the bovine Leydig cell.     

Age-related accumulation of lysosomes and other cytological features in active thyroid follicles of the CBA mouse

Summary This study attempts to elucidate the mechanism through which lysosomal accumulation occurs with age in the epithelial cells of the thyroid gland and especially in the active follicles of the aging mouse thyroid. Thyroid morphology and function in old CBA (at least 24 months of age) male mice were compared with those in young (2 months of age) animals. The effects of different intake of iodine were tested and compared in both cohorts, each of which was divided into three groups: (i) low iodine group, (ii) moderate iodine group, and (iii) high iodine group. As expected, the present work confirmed the well-known accumulation with age of cold follicles coexisting with active follicles in the old mouse thyroid. Attention has been focused on the active follicles whose follicular cells contained in their cytoplasm a large number of pleomorphic dense bodies. The lysosomal nature of these bodies, referred to as secondary lysosomes, was confirmed by histochemistry; however, they displayed variability in acid phosphatase staining. In old animals, regardless of the type of iodine regimen, the ratio between relative follicular volume and relative colloid volume as determined by morphometry remained unchanged. Ultrastructurally, the relative volume occupied by secondary lysosomes in active follicles was always higher than in the young groups. Autoradiographic studies with ¹²⁵I revealed that a large part of the radioactivity was located in secondary lysosomes of thyroid cells in active follicels of old mice when radioiodine was injected 3 weeks before death. Two different types of vacuoles were present in a non-negligible number of thyrocytes of the active follicles in aged cohorts. The first type was made up of grossly dilated rough endoplasmic cisternae, the second corresponded to intracytoplasmic microfollicular vacuoles. Both aspects have been described in conditions of chronic stimulation. It is concluded (1) that different intake of iodine for 6 weeks does not modulate the thyroid morphology in old mice; (2) that in the thyrocytes of the active follicles in old mice accumulation of secondary lysosomes occurs due to a slowdown of turnover; and (3) that the follicular cells of active follicles feature morphological aspects suggesting a hyperactive state compensating the lack of hormone production in the cold follicles.     

Characterization of auto-regulation of the human cardiac α1 subunit of the L-type calcium channel: Importance of the C-terminus

The carboxyl terminal of the L-type calcium channel _1C subunit comprises approximately one third of the primary structure of the ₁ subunit (> 700 amino acids residues). This region is sensitive to limited posttranslational processing. In heart and brain the _1C subunits are found to be truncated but the C-terminal domain remains functionally present. Based on our previous data we hypothesized that the distal C-terminus (approximately residues 1650–1950) harbors an important, predominantly inhibitory domain. We generated C-terminal-truncated _1C mutants, and after expressing them in combination with a ₃ subunit in HEK-293 cells, electrophysiological experiments were carried out. In order to dissect the important inhibitory part of the C-terminus, trypsin was dialyzed into the cells. The data provide evidence that there are multiple residues within the inhibitory domain that are crucial to the inhibitory process as well as to the enhancement of expressed current by intracellular application of proteases. In addition, the expression of the chimeric mutant _1C1673-DRK₁ demonstrated that the C-terminal is specific for the heart channel.     

Intercellular junctions in nerve-free hydra

Summary Epithelial cells of nerve-free hydra contain septate and gap junctions. In thin sections the gap junctions are characterized by a gap of 3–4 nm. Freeze-fracture demonstrates the presence of septate junctions and two further types of structures: (i) the E-type or inverted gap junctions with particles in an en plaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice constant of 8 nm. We propose that these structures are also gap junctions.     

On the localization,the fine structure,and the chemical composition of crystalline inclusions in theThesium humifusum haustoria

Summary TheThesium humifusum haustoria onMedicago marina roots, fixed in October–November, frequently contain in the cytoplasm of their cells inclusions consisting of sticks 0,3 m in thickness and 8 to 10 m in lenght, alone or associated in stacks of 3 or 6 units. These sticks consist of fibres, 10 nm in thickness, oriented in the same direction and separated from the others by a gap of 8 nm; these fibres seem to be composed of helically wound filaments and a less electron-dense matrix.The chemical composition of these inclusions was studied by enzymatic digestion in ultra thin sections; pronase digested the cytoplasmic paracrystals. This demonstrates that they are composed primarily of protein.The physiological significance of the inclusions is discussed: presumably the haustorium functions as a storage organe.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

On the fine structure of the aglomerular renal tubule in Lophius piscatorius

Summary The fine structure of the secretory tubules in the kidney of the aglomerular goose-fish (Lophius piscatorius) is described. The cells have a pyramidal shape, are joined together by multiple desmosomes, and share as main characteristics: abundant and deep inflections of the basal and lateral cell membranes; coated luminal plasma membranes forming multiple microvilli or a genuine brush border; moderate numbers of comparatively small mitochondria, usually unassociated with the basal and lateral plasma membrane specializations; numerous multivesicular bodies occuring in the apical cytoplasm; abundant large lysosome-like bodies in the intermediate regions of the cytoplasm; and comparatively poor development of endoplasmic reticulum and Golgi apparatus.The observations suggest that the cells perform both absorptive and secretory functions and are metabolically unusually active in autolytic and heterolytic work. Comparisons with other aglomerular species indicate that the ability for active secretory function is not necessarily dependent on a close association between plasma membrane and mitochondria; however, this ability does appear to require a markedly increased basal and/or lateral cell surface created by multiple invaginations of the plasma membrane. The abundance of desmosomes and associated structures appears to represent a unique structural specialization of the goosefish tubule, and indicates that the cells must be firmly anchored to one another to supply a rigid and mechanically continuous lining of the tubule. The multivesicular bodies probably represent endocytic vacuoles which fuse with apical vesicles and invaginate their outer membrane to form the internal vesicles; they appear to transform to ambilysosomes via a function as heterophagosomes and — later — combined hetero- and autophagosomes.Supported by grants from Karolinska Institutet, Fonden til Videnskabens Fremme and Konsul Johannes Fogh-Nielsen og fru Ella Fogh-Nielsens Légat. Part of the study was performed at the Zoological Station at Naples, Italy. The assistance of Mrs. Britt-Marie Karlsson is gratefully acknowledged.     

Potassium permanganate fixative and the electron microscopy of sieve tube contents

Summary Sieve tube slime is probably fibrillar as suggested by other workers. The granular material seen in electron micrographs of sieve tubes fixed with potassium permanganate is mostly a precipitate produced by the reaction of the permanganate with the sieve tube contents, particularly with sucrose, and with reagents used in the fixing, dehydrating and embedding process. Potassium permanganate is not therefore a good fixative for the electron microscopy of sieve tube contents. Neither is it suitable for the study of fine structure in vacuoles or vesicles containing precipitable solutes in other kinds of cells nor for examining the fine structure of their ground cytoplasm.     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.     

2-aminopurine induced DNA repair in E. coli

Summary The survival of UV-irradiated phages is increased when host bacteria are grown in the presence of the base analogue 2-aminopurine (2AP) before infection. This increase in survival, which we have called 2AP-reactivation depends upon the concentration of 2AP and the time of exposure to 2AP. 2AP-reactivation can be distinguished from Weigle-reactivation in that it is not accompanied by an increase in mutagenesis, does not act on the single-stranded DNA bacteriophage X174, and occurs in recA and lexA bacteria. 2AP reactivation does not appear to involve known systems of recombinational repair, as it occurs in recB and recF bacteria, or excision repair, as it occurs in uvrA and uvrB bacteria. It is however dependent upon DNA polymerase I.     

PEG-enhanced,electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts

Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such novel cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm^–1 for 50 s each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These novel grape cells remained viable for several hours.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichloro-phenoxyacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-amino-ethyl ether)-N,N,N,N - MES 2-[N-Morpholino]ethanesulfonic acid - MOPS 3-[N-Morpholino]propanesulfonic acid - PVP polyvinylpyrrolidone     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels

Summary The observation of secretory phenomena in mouse thymic epithelial cells is disappointing since no real secretion image is found. An adequate technique for such a study is to block the secretion pathway and to observe by electron microscopy cells accumulating secretory products. For this purpose, we used three means of blocking secretion: Firstly, since the thymic epithelial cell is regulated by a feedback phenomenon, secretion was blocked by antibodies against thymulin, one of the hormones secreted by these cells. Secondly, colchicine was used to modify the intracellular transport of the secretory product. In both of these types of experiments, electron microscopy showed a great increase in the number of clear vacuoles and their granular contents in epithelial cells. In a third series of experiments, we used monensin at a concentration that blocks the intracellular transport of secretory proteins at the various levels of the Golgi apparatus. In this series, only an increased number of vacuoles was observed, but they appeared devoid of all granular content. It can be concluded that in the thymic epithelial cell, a discrete system of secretion directs the passage of the product, originating in the cisternae of the endoplasmic reticulum, into clear vacuoles, the terminal element of the cellular secretory apparatus.