Taenia solium: cell reactions to the larva (Cysticercus cellulosae) in naturally parasitized, immunized hogs

In hogs naturally infected with Taenia solium larvae (i.e., Cysticercus cellulosae), we studied the host response induced by antigens obtained from the larvae. Histopathological studies of cysticerci removed after 4 and 8 weeks of immunization showed an intense inflammatory reaction surrounding the larvae. The response was greater in the 8-week specimens. A dense layer of eosinophils was in close contact with the external membrane of the bladder wall and, in several cases, the eosinophils had infiltrated this tegument. Many eosinophils were seen in the spiral canal of larvae. This infiltration by eosinophils increased with time. Preparations from the 8-week samples showed many degenerated and disrupted eosinophils whose granules were found in close contact with the outer membrane of the larval tegument and, in some cases, had entered through the broken surface of this structure. More than 90% of the larvae were found in various stages of degeneration; the rest were completely destroyed and surrounded by a mass of eosinophils. After immunization, peripheral blood eosinophilia increased to 17%, whereas the eosinophilia of the control hog was 4% throughout the study. The larval worms removed from control hogs showed intact structures, with a low degree of infiltration by eosinophils and a discrete inflammatory reaction surrounding the bladder wall of the larvae.     

Cellular localization of 3H-oestradiol in the hypothalamus

Summary The hypothalamus of male and female rats, given 0.3 g/100 g body weight of 6.7-³H-oestradiol-17 and killed 1 hour after the injection, was examined by autoradiography in order to 1) localize the areas and the cells involved in the uptake of the hormone, and 2) study the intracellular localization of the labelled material.Only nerve cells contained radioactive material while glial and ependymal cells were not significantly labelled. In the anterior hypothalamus, labelled nerve cells were concentrated in areas corresponding to nucleus preopticus medialis and nucleus preopticus, pars suprachiasmatica. The nucleus supraopticus was unlabelled. In the medial basal hypothalamus, neurons corresponding to the nucleus arcuatus and the lateral part of the nucleus ventromedialis showed marked labelling. No significant labelling was observed in the nucleus paraventricularis, pars magnocellularis.Although the individual nerve cells varied in their extent of labelling, the major proportion of the silver grains were consistently concentrated over the nuclei. Castration was not found to influence the results. The findings were essentially the same in male and female rats and appear to suggest that oestradiol exerts a direct effect on nerve cells in certain hypothalamic areas.This work was supported by grants from the Norwegian Cancer Society, Nordisk Insulinfond and Anders Jahres Fond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Microelectrode-recorded development of the symplasmic autonomy of the sieve element/companion cell complex in the stem phloem of Lupinus luteus L.

From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.Abbreviations LYCH Lucifer Yellow CH - membrane potential - PMF proton-motive force - PP phloem parenchyma cell - SE/CC-complex sieve element/companion cell complex - SR-G sulphorhodamine G     

Cellular location,activity states,and macromolecular organization of glucoamylase in Clostridium thermosaccharolyticum

By application of immunocytochemical techniques at the electron microscope level, glucoamylase was localized to the cell periphery in Clostridium thermosaccharolyticum during and following growth on starch, sucrose or glucose. Levels of immunolabelling were found to be relatively independent of growth substrate and of phase of growth, whereas previous studies had demonstrated strong dependence of glucoamylase activity on growth conditions; previously high levels of glucoamylase activity had been detected after growth on starch (i.e. during the stationary phase after growth) and only very low activities detected during exponential growth and following growth on glucose. The results presented demonstrate that levels of the glucoamylase protein are independent of measurable enzyme activity, and imply that the protein is constitutive. This indicates that the protein can exist in active and inactive states in the cell. By analogy with similar systems, we consider it likely that maturation or activation of newly synthesized glucoamylase occurs during (or following) transport through the cytoplasmic membrane. Electron microscopy of individual protein molecules which had been subjected to negative staining revealed that the enzyme consists of two domains of approximately equal size which are linked by a hinge region.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

Phylogenetic position of Dictyostelium inferred from multiple protein data sets

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.Correspondence to: T. Miyata     

Nanomolar Amyloid β Protein-Induced Inhibition of Cellular Redox Activity in Cultured Astrocytes

Abstract: It has been previously reported that Alzheimer's amyloid β protein (Aβ) induces reactive astrocytosis in culture. In the present study, we found that Aβ potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of Aβ on astrocytes. First, Aβ-induced reactive morphological change was suppressed by the presence of serum or thrombin, and Aβ inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 µ M or more) were required for Aβ to induce reactive astrocytosis, whereas nanomolar concentrations (0.1–100 n M ) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar Aβ was virtually irreversible, but nanomolar Aβ-induced inhibition of cellular redox activity was reversed by washing out Aβ. Furthermore, as it has been reported that Aβ neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to Aβ. However, neither Aβ-induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.