Experimental amebiasis: immunohistochemical study of immune cell populations

Distribution of immune cell populations was studied in a C3H/HeJ mouse model of intestinal amebiasis from 5 to 60 days post-inoculation with Entamoeba histolytica, using immunoperoxidase techniques. At various time intervals, the ceca from mice were fixed in 10% formalin, dehydrated, embedded and sectioned at 5 microm. Sections were incubated with conjugated peroxidase-labelled antibodies to mouse IgA, IgM, and IgG. Color was developed with 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 solution. CD3, CD4, and CD8 cells, as well as neutrophils were detected by reacting with biotin-conjugated anti-mouse CD3, CD4, CD8, and CD11 monoclonal antibodies, followed by their incubation with avidin-peroxidase and color development with DAB/H2O2 solution. Erythrocin B and toluidine blue were used to stain eosinophils and Mast cells, respectively. It was observed that the IgA+ plasma cell was the dominating immune cell present in the mucosa, although eosinophils, neutrophils, CD3+, CD4+, CD8+, IgM+, IgG+ cells and Mast cells were also seen. Results of this study suggest that infiltration of immune cells at the mucosal surface during intestinal amebiasis might be important in the defense against this parasite.     

Can dog-ear formation be decreased when an S-shaped skin resection is used instead of a spindle skin resection? A three-dimensional analysis of skin surgery techniques using the finite element method

Dog-ear formation is often unavoidable with resection and suturing of the skin, including spindle excision. Regarding dog-ear formation after basic spindle skin resection during removal of a round tumor of the skin, we quantitatively analyzed the frequency of dog-ear formation with respect to the following three techniques: previous spindle skin resection, S-shaped skin resection, which has been experientially considered to induce limited deformity, and mosque-shaped skin resection for control. To date, by using paper models or sponges, various techniques of skin resection have been simulated in the field of plastic surgery. In the present study, we performed three-dimensional simulation and analyzed three different techniques of skin resection by using the finite element method. As a result, image simulation demonstrated that the frequency of dog-ear formation was limited by S-shaped, spindle, and mosque-shaped skin resection, in descending order.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Amino acid conditions near the GPI anchor attachment site of prion protein for the conversion and the GPI anchoring

Prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored protein, and the C-terminal GPI anchor signal sequence (GPI-SS) of PrP is cleaved before GPI anchoring. However, mutations near the GPI anchor attachment site (the ω site) in the GPI-SS have been recognized in human genetic prion diseases. Moreover, the ω site of PrP has not been identified except hamster, though it is known that amino acid restrictions are very severe at the ω and ω + 2 sites in other GPI-anchored proteins. To investigate the effect of mutations near the ω site of PrP on the conversion and the GPI anchoring, and to discover the ω site of murine PrP, we systematically created mutant murine PrP with all possible single amino acid substitutions at every amino acid residue from codon 228 to 240. We transfected them into scrapie-infected mouse neuroblastoma cells and examined the conversion efficiencies and the GPI anchoring of each mutant PrP. Mutations near the ω site altered the conversion efficiencies and the GPI anchoring efficiencies. Especially, amino acid restrictions for the conversion and the GPI anchoring were severe at codons 230 and 232 in murine PrP, though they were less severe than in other GPI-anchored proteins. Only the mutant PrPs presented on a cell surface via a GPI anchor were conversion competent. The present study shows that mutations in the GPI-SS can affect the GPI anchoring and the conversion efficiency of PrP. We clarified for the first time the ω site of murine PrP and the amino acid conditions near the ω site for the conversion as well as GPI anchoring.     

Cross-sequence transmission of sporadic Creutzfeldt-Jakob disease creates a new prion strain

The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of abnormal isoform of PrP (PrP(Sc)) are major determinants of the clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Here we found that the transmission of sCJD prions from a patient with valine homozygosity (129V/V) and type 2 PrP(Sc) (sCJD-VV2 prions) to mice expressing human PrP with methionine homozygosity (129M/M) generated unusual PrP(Sc) intermediate in size between type 1 and type 2. The intermediate type PrP(Sc) was seen in all examined dura mater graft-associated CJD cases with 129M/M and plaque-type PrP deposits (p-dCJD). p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology, and the identical type of PrP(Sc) when inoculated into PrP-humanized mice with 129M/M or 129V/V. These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions.     

Reactive oxygen species trigger ischemic and pharmacological postconditioning: in vivo and in vitro characterization

Reactive oxygen species (ROS) generated by ischemic and pharmacological preconditioning are known to act as triggers of cardiac protection; however, the involvement of ROS in ischemic and pharmacological postconditioning (PostC) in vivo and in vitro is unknown. We tested the hypothesis that ROS are involved in PostC in the mouse heart in vivo and in the isolated adult cardiac myocyte (ACM). Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion with or without ischemic or pharmacologic PostC (three cycles of 20 s reperfusion/ischemia; 1.4% isoflurane; 10 mg/kg SNC-121). Additional groups were treated with 2-mercaptopropionyl glycine (MPG), a ROS scavenger, 10 min before or after the PostC stimuli. Ischemia-, isoflurane-, and SNC-121- induced PostC reduced infarct size (24.1+/-3.2, 15.7+/-2.6, 24.9+/-2.6%, p<0.05, respectively) compared to the control group (43.4+/-3.3%). These cardiac protective effects were abolished by MPG when administered before (40.0+/-3.6, 39.3+/-3.1, 38.5+/-1.6%, respectively), but not after the PostC stimuli (26.6+/-2.3, 17.0+/-2.2, 23.9+/-1.7%, respectively). Additionally, ACM were subjected to a simulated ischemia/reperfusion protocol with isoflurane and SNC PostC. Isoflurane- and SNC-induced PostC in vitro were abolished by prior treatment with MPG. These data indicate that ROS signaling is an essential trigger of ischemic and pharmacological PostC and this is occurring at the level of the cardiac myocyte.     

Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death

Senescence or cell death in plant leaves is known to be inducible by darkness or H(2)O(2). When the Arabidopsis gene MAX2/ORE9 is disrupted, leaf senescence or cell death in response to the above stimuli is delayed. Because the rice (Oryza sativa L.) gene DWARF3 (D3) is orthologous to MAX2/ORE9, we wished to know whether disruption of D3 also results in increased longevity in leaves. We found that darkness-induced senescence or H(2)O(2)-induced cell death in the third leaf [as measured by chlorophyll degradation, membrane ion leakage and expression of senescence-associated genes (SAGs)] in a d3 rice mutant was delayed by 1-3 d compared to that in its reference line Shiokari. Moreover, the mRNA levels of D3, HTD1 and D10, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and MAX4, respectively, increased during cell death. These results suggest that D3 protein in rice, like MAX2/ORE9 in Arabidopsis, is involved in leaf senescence or cell death.     

Enhancement of Sodium Current in NG108-15 Cells during Neural Differentiation is Mainly due to an Increase in NaV1.7 Expression

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma × glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10⁻⁷ M tetrodotoxin (TTX). (2) The only Na_V mRNA with an increased expression level during neuronal differentiation was that for Na_V1.7, as observed by real-time PCR analysis. (3) The increase in the level of Na_V1.7 α subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of Na_V1.7 α subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na⁺ channel, Na_V1.7.