Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

A comparative study of the biological properties of venoms of some old world vipers (subfamily viperinae).

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 30 samples of venoms from nine species (12 taxa) of the old world vipers (Subfamily Viperinae) including snakes from the genera Bitis, Causus, Cerastes, Echis, Eristicophis and Pseudocerastes, were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. Examination of the biological properties of the venoms of the Viperinae tested indicates the presence of common venom biological characteristics at the various phylogenic levels. 3. Venoms of most species of the Viperinae examined exhibited characteristic biological properties at the species level, and this allows the differentiation of the Viperinae species by differences in their biological properties. 4. Particularly useful for this purpose, are the effects of venom on kaolin-cephalin clotting time of platelet poor rabbit plasma and the Sephadex G-75 gel filtration pattern and arginine ester hydrolase activity of the venom.     

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.     

Plasmodium falciparum: analysis of karyotype polymorphism using chromosome-specific probes

Chromosome size variation in Plasmodium falciparum has been examined using a double heterogenous pulse field gradient electrophoresis apparatus and a series of chromosome-specific probes. In the 11 different isolates analyzed the chromosomal markers always hybridized to the corresponding chromosome, indicating that translocations do not significantly contribute to chromosome size variations. Furthermore, despite probes specific for chromosomes 5 and 6 no evidence was obtained to support the hypothesis of a chromosome duplication involving these chromosomes. The double heterogenous electric field combined with longer pulse times allowed the genome to be resolved into a larger number of chromosomal bands and as a result permitted the more precise mapping of cloned genes.     

A comparative study of the biological activities of venoms from snakes of the genus Agkistrodon (moccasins and copperheads)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon (A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. Venoms of the subspecies of A. contortrix and A. piscivorus do not differ significantly in their biological activities.     

A comparative study of the biological properties of Australian elapid venoms

1. The hemorrhagic, procoagulant, anticoagulant, protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, arginine ester hydrolase, phospholipase A, 5'-nucleotidase and hyaluronidase activities of 39 samples of venoms from 13 species (15 taxa) of Australian elapids were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate that Australian elapid venoms can be divided into two groups: procoagulant Australian venoms (including N. scutatus, N. ater, O. scutellatus, O. microlepidotus, P. porphyriacus, T. carinatus, H. stephensii and P. textilis) and non-procoagulant Australian venoms (including A. superbus, P. colletti, P. australis, P. guttatus and A. antarcticus). 3. The non-procoagulant Australian venoms exhibited biological properties similar to other elapid venoms, while the procoagulant Australian venoms exhibited some properties characteristic of viperid venoms. 4. The data show that information on venom biological properties can be used for differentiation of many species of Australian elapids. 5. Particularly useful for this purpose are the hyaluronidase, alkaline phosphomonoesterase, acetylcholinesterase, and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.     

Malignant gastrointestinal stromal tumor presenting with hemoperitoneum in puerperium: report of a case with review of the literature

Background

Leiomysarcoma of intravascular origin is an exceedingly rare entity of malignant soft tissue tumors. They are most frequently encountered in the retroperitoneum arising from the inferior vena cava and are scarcely found to arise from vessels of the extremities. These tumors were analysed with particular reference to treatment outcome and prognosis. The aim of this article is to broaden the knowledge of the clinical course of this rare malignancy.

Method

During 2000 and 2009 twelve patients were identified with an intravascular origin of a leiomyosarcoma. Details regarding the clinical course, follow-up and outcome were assessed with focus on patient survival, tumor relapse and metastases and treatment outcome. 3 year survival probability was calculated using Kaplan-Meier method.

Results

Vascular leiomyosarcomas accounted for 0.7% of all malignant soft tissue tumors treated at our soft tissue sarcoma reference center. The mean follow up period was 38 months. Tumor relapse was encountered in six patients. 6 patients developed metastatic disease. The three year survival was 57%.

Conclusion

Vascular leiomysarcoma is a rare but aggressive tumor entity with a high rate of local recurrence and metastasis.     

Proteomic analysis of zebrafish (Danio rerio) embryos exposed to cyclosporine A

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes.     

Plasmodium species: flow cytometry and microfluorometry assessments of DNA content and synthesis

Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C50 value of 5 microM of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content.