Neurosecretion. XIII. The ultrastructure of the corpus cardiacum of the insect Leucophaea maderae

Summary The corpora cardiaca of Leucophaea maderae contain two classes of intrinsic elements, parenchymal and interstitial cells. The parenchymal cells produce a secretory material first visible in the Golgi zones of the perikarya in the form of distinct electron-opaque granules. These undergo changes (gradual loss of electron-density, emergence of internal structure) as they accumulate in cellular processes.The parenchymal cells are best classified as neuroglandular elements since, in addition to secretory inclusions, they possess characteristics of ganglion cells such as axonlike processes, neurotubules, and sheaths. These covers are provided by branches of the second type of intrinsic elements, the interstitial cells. They are non-glandular structures of considerable morphological complexity. In the manner of glial elements, they permeate the entire organ and encapsulate not only the perikarya of parenchymal cells but cellular processes as well.The cytoplasmic processes include a) relatively short ones belonging to parenchymal cells, and b) long axons whose cell bodies lie within the central nervous system. Many of the latter contain electron-opaque granules of the kind found in electron-micrographs of typical neurosecretory cells. These extrinsic granules represent the second category of secretory products stored within the corpora cardiaca. By comparison with the product of the intrinsic gland cells, the neurosecretory granules seem to be fairly stable. Neither type seems to pass through the connective tissue sheath of the corpus cardiacum in the form of discrete granular entities.This sheath, which sends branches into the interior of the corpora cardiaca, has the properties of a basement membrane. It represents a pathway for the exchange of substances between the cellular components of the corpus cardiacum and the surrounding hemocoele.The dual character of the corpus cardiacum, namely that of a storage and release center for extrinsic neurosecretory substances and of an endocrine organ in its own right, is herewith established beyond doubt. The number of secretory products discernible on the basis of their morphology and localization (two size categories of extrinsic and one intrinsic type of granules) does not match the variety of physiologically active principles known at present. The assignment of specific functions to discrete morphological elements must await further studies.Supported by Research Grants A-3984 and B-2145 from the U.S.P.H.S.     

The plant nucleus in mycorrhizal roots: positional and structural modifications

Summary— Positional and structural modifications were demonstrated in nuclei of leek cells, after establishment of a symbiosis with two vesicular-arbuscular fungi, Glomus versiforme and Glomus E₃. By combining light, immuno-electron microscopy and morphometry, the fungi were shown to have a direct effect on the host nuclear morphology: the effect was confined to a specific plant tissue (the cortical parenchyma) and to a moment of the fungal morphogenesis (the arbuscule). When they branch to form the complex structures called arbuscules in the inner parenchyma cells, the host nucleus migrates from the periphery of these cells towards their centre. In addition, it becomes larger and lobed, with a decondensed chromatin. A monoclonal antibody that mostly binds to the condensed chromatin revealed a significant decrease in gold labelling intensity over the nuclei of the colonized cells. These modifications suggest that the nuclear migration and the changes in chromatin organization are related to the modifications in gene expression observed during the establishment of mycorrhizal symbiosis.     

Characterization of a cell wall β-galactosidase of Cicer arietinum epicotyls involved in cell wall autolysis

β-Galactosidase (EC 3.2.1.23) has been established as the main enzyme involved in the autolytic process. The enzyme extracted from cell walls of epicotyls of Cicer arietinum L. cv. Castellana with 3 M LiCl is a 45 kDa protein composed of a single subunit, having an optimum pH of 4; an optimum temperature of 45°C and K_m and V_max of 1.72 m M and 18.5 nkat (mg protein)^–1 respectively, as evaluated against p -nitrophenyl-β- d -galactopyranoside. The enzyme is inhibited by Hg²⁺, d -galactono-1,4-lactone and galactose, substances that also inhibit the autolytic process. Ca²⁺ and EDTA, which do not affect the activity of the β-gaiactosidase, do however modify the hydrolysis of the cell wall mediated by the enzyme, and they also inhibit the autolytic process. Ca²⁺ decreased both processes, whereas EDTA increased them; and when both substances were added together, their individual effects were neutralized. The effects of both agents is probably due to modifications in the cell wall that prevent access of the enzyme to its substrate.     

Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata

In marine benthic invertebrates with complex life cycles, recruitment success, juvenile survival, and growth may be affected by variation in both maternal factors and environmental conditions prevailing during preceding embryonic or larval development. In an estuarine crab, Chasmagnathus granulata, previous investigations have shown that initial larval biomass is positively correlated with the biomass of recently extruded eggs, and it depends also on the salinity experienced during embryogenesis. Biomass at hatching has consequences for the subsequent larval development which, in this species, comprises two alternative developmental pathways with four or five zoeal instars (short or long pathway) and a megalopa. Larvae hatching with a lower than average biomass tend to develop through the long pathway and metamorphose to megalopae with higher biomass. In the present study, we show experimentally that the long pathway produces also significantly larger juveniles (crab size measured as carapace width, biomass as dry mass, carbon and nitrogen contents). Compared with juveniles originating from the short pathway, those from the long pathway showed in successive instars longer moulting cycles and larger carapace width, but lower size increments at ecdysis. In consequence, differences in size or biomass of long pathway vs short pathway crabs tended to disappear in later instars (after stage V). Furthermore, we tested in juveniles the tolerance of starvation at three salinities (5‰, 15‰, 32‰). Tolerance of starvation was significantly higher in juveniles originating from the long pathway, indicating higher energy reserves. While salinity played only a minor role for survival, it exerted significant effects on the time of moulting to the second juvenile instar, regardless of the preceding developmental pathway. The biomass of first juveniles obtained from the short pathway showed a significant positive correlation with the biomass of the freshly hatched zoea I, but not in those from the long pathway. In conclusion, the fitness of juvenile C. granulata is linked with previous developmental processes and environmental conditions during the embryonic and larval phase. Hence, a better understanding and prediction of the recruitment success of marine benthic invertebrates with a complex life cycle may require more comprehensive life‐history investigations.     

Characterization of xanthans from selected Xanthomonas strains cultivated under constant dissolved oxygen

Seventeen wild-type Xanthomonas isolates were screened in terms of broth viscosity in shake-flasks. As culture conditions affect polymer characteristics, a fair comparison among isolates required their cultivation in a fermenter under controlled dissolved oxygen tension. Three isolates and a reference strain were studied. The mean molecular weights and molecular weight distributions of their xanthans were determined. Products showed different pyruvate (0.2–7%), acetate (5–10%) and proteinaceous nitrogen (1–3%) contents. The selected isolates exhibit properties which could improve xanthan gum production and some could be used to produce polymers with specific characteristics.     

Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.     

Application of immunoprecipitation techniques to the diagnosis of cutaneous and extracutaneous forms of sporotrichosis

Sporotrichosis is a mycosis which in our country has been described as only producing cutaneous and subcutaneous infections; in other countries it has been reported as causing pulmonary, bone, joint and meningeal infections.Possibly the systemic form also occurs in Venezuela since, along with the causative agent, all other factors considered as predisposing to systemic infection exist here.Through immunodiffusion (ID) and immunoelectrophoresis (IEP) and using a filamentous form metabolic antigen from Sporothrix schenckii, we were able to demonstrate precipitating antibodies against this fungus both in patients with cutaneous sporotrichosis, diagnosed by culture of the infecting agent, and in patients with pulmonary lesions. The IEP test showed the presence of an anodic arc which we have called the S arc, which could be one of the specific antigens of S. schenckii.

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Resumen En Venezuela, el diagnóstico de la esporotricosis solo se plantea ante lesiones cutáneas y subcutáneas, sin embargo, no deja de llamar la atención que siendo esta micosis la más frecuente en nuestro medio y existiendo concomitantemente en la población todos los factores predisponentes para que el S. schenckii invada órganos internos, estas localizaciones no han sido descritas.La incorporación del antígeno del S. schenckii para detectar anticuerpos circulantes en la ID en nuestros estudios serológicos de rutina, nos permitió observar la presencia de bandas de precipitación contra este antígeno en sueros de pacientes con lesiones pulmonares, igualmente fueron detectados anticuerpos en pacientes con esporotricosis cutáneas diagnosticadas por cultivo.El estudio de la estructura antigénica de este hongo, puso de manifiesto la presencia de un arco de localización anódica, que podría ser producido por uno de los antígenos específicos de este hongo; este arco que denominamos S fue visualizado en todos los casos positivos de esporotricosis cutáneas, al ser estudiados sus sueros en la IEF.Los estudios realizados en sueros de pacientes con lesiones pulmonares pusieron de manifiesto anticuerpos precipitantes en la ID e IEF, con las caractéristicas de evidenciar más de una línea de precipitación en la ID y un arco S más largo en la IEF, pudiendo ser esto originado por mayor producción de anticuerpos circulantes. Se plantea la necesidad de obtener el cultivo del S. schenckii de las posibles lesiones extracutáneas de esta micosis.

     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Neurosecretion XVII. Experimentally induced release of neurosecretory material by exocytosis in the insect Leucophaea maderae

Summary In the corpora cardiaca of the insect Leucophaea the administration of serotonin elicits ultrastructural features indicative of the extrusion of neurosecretory material by exocytosis. The response to the stimulus and the process of extrusion seem to occur at considerable speed. Nearly all of the 30 test animals, fixed at various intervals starting as early as 3 min after the injection of the drug, show granules captured at the moment of leaving the axon as well as fully exteriorized secretory material. The fact that many of these granules are much smaller than the typical neurosecretory type speaks for intracellular fragmentation of the latter prior to the discharge of this cellular product. After 25 min or more the extruded electron dense structures show signs of breakdown. The apparent speed of these phenomena accounts for the dearth of omega-type configurations observed in unstimulated specimens of this species. The possible relationship between the membrane phenomena involved in exocytosis and the transient protrusions of bounding membranes of neurosecretory granules described in earlier papers remains to be clarified.Supported by N.S.F. research grant BMS 74-12456