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.     

Induction of diabetes by PolyI:C and anti-RT6.1 antibody treatment in DR-BB rats.

To determine whether environmental factors could affect the incidence of diabetes in RT6.1+ lymphocytes-depleted diabetes resistant (DR) BB rats, we tested polyinosinic-polycytidylic acid (Poly I:C), as an immune activator, in conjunction with anti-RT6.1 antibody in DR-BB rats which were bred in a specific pathogen free (SPF) condition. Diabetes was induced by the combined administration of poly I:C and anti-RT6.1 antibody. The use of poly I:C or anti-RT6.1 antibody alone did not cause diabetes. These results suggest that RT6.1+ T lymphocytes regulate autoimmune diabetes and that non-specific immune activation caused by environmental factors plays a key role in inducing diabetes in DR-BB rats.     

Ciliate protozoa in the rumen of Kafue lechwe, Kobus leche kafuensis, in Zambia, with the description of four new species.

The composition of the rumen ciliate fauna in 76 Kafue lechwe inhabiting a limited area in Zambia was surveyed and five genera containing 24 species with 16 formae belonging to the family Ophryoscolecidae were identified. Four new species belonging to Diplodiniinae were recognized and described as Diplodinium lochinvarense n. sp., Diplodinium leche n. sp., Diplodinium zambiense n. sp., and Metadinium ossiculi n. sp. In addition, Ostracodinium gracile form fissilaminatum Dogiel, 1932 was found for the second time and described as Metadinium fissilaminatum n. comb. The species composition was fairly unusual. Seven of the species have been found only in African wild antelopes and these species were found more frequently than cosmopolitan species. There was no evidence of isotrichid species. The average density of ciliates per 1 ml of the rumen fluid was 25.7 x 10(4), and the number of ciliate species per head of host was 10.8.     

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

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.     

Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we isolated primary osteoblasts derived from transgenic rats harboring a dominant negative ER mutant, rat ERalpha (1-535) cDNA, and from their wild-type littermates. We observed that the rate of cell growth of osteoblasts from the transgenic rats was reduced compared to that of wild-type osteoblasts. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. D-type cyclins including cyclin D1, cyclin D2, and cyclin D3 are cell cycle regulators that promote progression through the early-to-mid G1 phase of the cell cycle. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Moreover, an anti-estrogen ICI 182,780 abolished the induction of the expression of D-type cyclins by 17beta-estradiol. Our findings indicate that estrogen and its receptors enhance Cdk4/6 activities through the induction of D-type cyclins, leading to the growth promotion of osteoblasts.     

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.