Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites. Target for the antimalarial activity of bestatin

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wildtype parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 mu M) compared with the parent parasites (EC50 25 mu M). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation ( FISH). Participating laboratories were blinded to these test results. Oestrogen receptor ( ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH ( kappa coefficient=1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good ( kappa coefficient=0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

Host-specificity of monogenean (platyhelminth) parasites: a role for anterior adhesive areas?

Monogeneans (flatworms) are among the most host-specific of parasites in general and may be the most host-specific of all fish parasites. Specificity, in terms of a restricted spatial distribution within an environment, is not unique to parasites and is displayed by some fungi, insects, birds, symbionts and pelagic larvae of free-living marine invertebrates. The nature of cues, how habitats are recognised and how interactions between partners are mediated and maintained is of interest across these diverse associations. We review some experiments that demonstrate important factors that contribute to host-specificity at the level of infective stages (larvae of oviparous monogeneans; juveniles of viviparous gyrodactylids) and adult parasites. Recent research on immune responses by fish to monogenean infections is considered. We emphasise the critical importance of host epidermis to the Monogenea. Monogeneans live on host epidermis, they live in its products (e.g. mucus), monopisthocotyleans feed on it, some of its products are attractants and it may be an inhospitable surface because of its immunological activity. We focus attention on fish but reference is made to amphibian hosts. We develop the concept for a potential role in host-speciality by the anterior adhesive areas, either the specialised tegument and/or anterior secretions produced by monogeneans for temporary but firm attachment during locomotion on host epithelial surfaces. Initial contact between the anterior adhesive areas of infective stages and host epidermis may serve two important purposes. (1) Appropriate sense organs or receptors on the parasite interact with a specific chemical or chemicals or with surface structures on host epidermis. (2) A specific but instant recognition or reaction occurs between component(s) of host mucus and the adhesive(s) secreted by monogeneans. The chemical composition of fish skin is known to be species-specific and our preliminary analysis of the chemistry of some monogenean adhesives indicates they are novel proteins that display some differences between parasite families and species. (C) 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Laminar degeneration of the frontal and temporal cortex in neuronal intermediate filament inclusion disease (NIFID): a study using a-internexin immunohistochemistry

Objective: To determine the laminar distribution of the pathological changes in the frontal and temporal lobe in neuronal intermediate filament inclusion disease (NIFID). Method: The distribution of the alpha-intenexin-positive neuronal cytoplasmic inclusions (NCI), surviving neurons, swollen achromatic neurons (SN) and glial cell nuclei was studied across the cortex in gyri of the frontal and temporal lobe in 10 cases of NIFID. Results: The distribution of the NCI was highly variable within different gyri, a peak in the upper cortex, a bimodal distribution with peaks of density in the upper and lower laminae, or no significant variation in density across the cortex. The surviving neurons were either bimodally distributed or exhibited no significant change in density across the cortex. The SN and glial cell nuclei were most abundant in the lower cortical laminae. In half of the gyri, variations in density of the NCI across the cortex were positively correlated with the SN. In some gyri, the surviving neurons were positively correlated with the SN and negatively correlated with the glial cell nuclei. In addition, the SN and glial cell nuclei were positively correlated in over half the gyri studied. Conclusion: The data suggest that frontal and temporal lobe degeneration in NIFID characterized by NCI, SN, neuronal loss and gliosis extends across the cortical laminae with considerable variation between cases and gyri. alpha-internexin-positive neurons in the upper laminae appear to be particularly vulnerable. The gliosis appears to be largely correlated with the appearance of SN and with neuronal loss and not related to the NCI.

Neuropathological changes in ten cases of neuronal intermediate filament inclusion disease (NIFID):A study using α-internexin immunohistochemistry and principal components analysis (PCA)

Ten cases of neuronal intermediate filament inclusion disease (NIFID) were studied quantitatively. The α-internexin positive neurofilament inclusions (NI) were most abundant in the motor cortex and CA sectors of the hippocampus. The densities of the NI and the swollen achromatic neurons (SN) were similar in laminae II/III and V/VI but glial cell density was greater in V/VI. The density of the NI was positively correlated with the SN and the glial cells. Principal components analysis (PCA) suggested that PC1 was associated with variation in neuronal loss in the frontal/temporal lobes and PC2 with neuronal loss in the frontal lobe and NI density in the parahippocampal gyrus. The data suggest: 1) frontal and temporal lobe degeneration in NIFID is associated with the widespread formation of NI and SN, 2) NI and SN affect cortical laminae II/III and V/VI, 3) the NI and SN affect closely related neuronal populations, and 4) variations in neuronal loss and in the density of NI were the most important sources of pathological heterogeneity. © Springer-Verlag 2005.

Identification, distribution, and probability of infection in sympatric gastropods by digenean parasites at Pa-hay-okee, Everglades National Park using molecules and morphology

In this study, I determined the identity, taxonomic placement, and distribution of digenetic trematodes parasitizing the snails Pomacea paludosa and Planorbella duryi at Pa-hay-okee, Everglades National Park. I also characterized temporal and geographic variation in the probability of parasite infection for these snails based on two years of sampling. Although studies indicate that digenean parasites may have important effects both on individual species and the structure of communities, there have been no studies of digenean parasitism on snails within the Everglades ecosystem. For example, the endangered Everglade Snail Kite, a specialist that feeds almost exclusively on Pomacea paludosa, and is known to be a definitive host of digenean parasites, may suffer direct and indirect effects from consumption of parasitized apple snails. Therefore, information on the diversity and abundance of parasites harbored in snail populations in the Everglades should be of considerable interest for management and conservation of wildlife. Juvenile digeneans (cercariae) representing 20 species were isolated from these two snails, representing a quadrupling of the number of species known. Species were characterized based on morphological, morphometric, and sequence data (18S rDNA, COI, and ITS). Species richness of shed cercariae from P. duryi was greater than P. paludosa, with 13 and 7 species respectively. These species represented 14 families. P. paludosa and P. duryi had no digenean species in common. Probability of digenean infection was higher for P. duryi than P. paludosa and adults showed a greater risk of infection than juveniles for both of these snails. Planorbella duryi showed variation in probability of infection between sampling sites and hydrological seasons. The number of unique combinations of multi-species infections was greatest among P. duryi individuals, while the overall percentage of multi-species infections was greatest in P. paludosa. Analyses of six frequently-observed multiple infections from P. duryi suggest the presence of negative interactions, positive interactions, and neutral associations between larval digeneans. These results should contribute to an understanding of the factors controlling the abundance and distribution of key species in the Everglades ecosystem and may in particular help in the management and recovery planning for the Everglade Snail Kite.

Stray dogs in Bucharest, and the political economy of Romanian socialism. From hunting the “enemies” of Stalinist economy, to poisoning urban parasites, and the rise of urban anarchy: 1950s-1990s.

My paper discusses three different ways in which stray dogs have been intertwined with ideologies of economic and urban development in Romania. I categorize results from archival and ethnographic research under three major time periods: early socialism, late socialism, and post-socialism. During early socialism stray dogs were seen to be damaging the soviet economy by killing species that humans could also hunt, like rabbits. During late socialism, stray dogs appeared as the enemies of the communist city, and the department of urban sanitation was given orders to poison dogs with strychnine. Finally, the increasing number of stray dogs in Bucharest after the collapse of communism was seen as a direct result of former communist demolitions, and was also taken as a sign of the collapsing state. Through such examples my paper discusses how the state and particular population groups have seen dogs as parts of an unwanted and dangerous nature, rather than a species that needs to be protected. I argue that distinctions of nature and culture have served discourses of civilization and the view of Bucharest as a model socialist, and then European city. Throughout my paper I juxtapose the treatment of stray dogs with other, more “valued” urban natures like the protection of parks, the wide-spread hobby of pigeon breeding during socialist years, the most recent debate on saving the rural area of Rosia Montana from non-environmentally friendly methods of gold extraction, and the current trend of healthy eating and living.