The Histopathology of the Infection of Tilapia rendalli and Hypostomus regani (Osteichthyes) by Lasidium Larvae of Anodontites trapesialis (Mollusca, Bivalvia)

It is described the histopathology of the infection of Tilapia rendalli (Osteichthyes, Perciformes, Cichlidae) and Hypostomus regani (Osteichthyes, Siluriformes, Loricariidae) by lasidium larvae of Anodontites trapesialis (Mollusca, Bivalvia, Mycetopodidae). The larvae were encysted within the epidermis of the host, being surrounded by a thin hyaline membrane, 3-6 µm thick, of parasite origin. A proliferative host cell reaction did not occur. The histopathology of the infection shows that the lesions induced by the parasites are minimal. However, the numerous small lesions produced by the release of the larvae may provide optimal conditions for the infection by opportunistic pathogens, namely fungus, which may eventually cause the death of the host.

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Final Report of the Review Group on Health Service Care Staff

The National Health Strategy “Quality and Fairness, Health System for You” states“ a key objective of the human resource framework is to develop and explicitly value staff at all levels of the health system. This in turn benefits service users.” The strategy explicitly states that one of its initiatives was “to introduce the grade of Health Care Assistant (HCA) as a member of healthcare teams to assist and support nurses and midwives. A national six month training programme for Health Care Assistants to commence at the end of November 2001. Seventeen pilot programmes to be delivered by the health services in conjunction with the Further Education Training Awards Council (FETAC)”.

Digit somatotopy in the human cerebellum: a 7T fMRI study.

The representation of the human body in the human cerebellum is still relatively unknown, compared to the well-studied homunculus in the primary somatosensory cortex. The investigation of the body representation in the cerebellum and its somatotopic organisation is complicated because of the relatively small dimensions of the cerebellum, compared to the cerebrum. Somatotopically organised whole-body homunculi have previously been reported in both humans and rats. However, whether individual digits are represented in the cerebellum in a somatotopically organised way is much less clear. In this study, the high spatial resolution and high sensitivity to the blood oxygenation level dependent (BOLD) signal of 7T fMRI were employed to study the BOLD responses in the human cerebellum to the stroking of the skin of individual digits, the hand and forearm. For the first time, a coarse somatotopic organisation of the digits, ordered from D1-D5, could be visualised in individual human subjects in both the anterior (lobule V) and the posterior (lobule VIII) lobes of the cerebellum using a somatosensory stimulus. The somatotopic gradient in lobule V was found consistently in the posterior to anterior direction, with the thumb most posterior, while the direction of the somatotopic gradient in lobule VIII differed between subjects. No somatotopic organisation was found in Crus I. A comparison of the digit patches with the hand patch revealed that the digit regions are completely covered by the hand region in both the anterior and posterior lobes of the cerebellum, in a non-somatotopic manner. These results demonstrate the promise of ultra-high field, high-resolution fMRI for studies of the cerebellum.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

The L162V polymorphism of the PPARA gene is associated with stage C of heart failure.

In human heart failure (HF) peroxisome proliferator-activated receptor alpha (PPAR alpha) is downregulated and consequently, the expression of genes involved in fatty acid oxidation repressed. The L162V (rs1800206) is a functional polymorphism of the human PPAR alpha gene (PPARA). In the present study we have investigated whether this polymorphism is associated with the development of stage C of HF.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Characterization and evolution of the novel gene family FAM90A in primates originated by multiple duplication and rearrangement events

Genomic plasticity of human chromosome 8p23.1 region is highly influenced by two groups of complex segmental duplications (SDs), termed REPD and REPP, that mediate different kinds of rearrangements. Part of the difficulty to explain the wide range of phenotypes associated with 8p23.1 rearrangements is that REPP and REPD are not yet well characterized, probably due to their polymorphic status. Here, we describe a novel primate-specific gene family, named FAM90A (family with sequence similarity 90), found within these SDs. According to the current human reference sequence assembly, the FAM90A family includes 24 members along 8p23.1 region plus a single member on chromosome 12p13.31, showing copy number variation (CNV) between individuals. These genes can be classified into subfamilies I and II, which differ in their upstream and 5′-untranslated region sequences, but both share the same open reading frame and are ubiquitously expressed. Sequence analysis and comparative fluorescence in situ hybridization studies showed that FAM90A subfamily II suffered a big expansion in the hominoid lineage, whereas subfamily I members were likely generated sometime around the divergence of orangutan and African great apes by a fusion process. In addition, the analysis of the Ka/Ks ratios provides evidence of functional constraint of some FAM90A genes in all species. The characterization of the FAM90A gene family contributes to a better understanding of the structural polymorphism of the human 8p23.1 region and constitutes a good example of how SDs, CNVs and rearrangements within themselves can promote the formation of new gene sequences with potential functional consequences.

The externalisation of the asylum function in the European Union

This paper aims to identify and assess the main items in the strategy followed by the EU and its member states on the externalisation of their asylum function. First, it analyses the European harmonisation of the return to safe third countries and to countries of first asylum, which is carried out by means of readmission agreements. Second, it refers to the strategies defined by the Hague and the Stockholm programs concerning the External Aspects of the European Union Asylum Policy, on the detention centres for illegal immigrants abroad, and on the proposals for delocalisation of asylum applications processing centres beyond the EU borders. Finally, this paper considers whether the strategy of externalisation of the function of asylum sometimes lacks legitimacy, and to what extent there is a fair balance between the interests of the states and the protection of the human rights of refugees and asylum seekers.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Rapid prototyping of compliant human aortic roots for assessment of valved stents.

Adequate in-vitro training in valved stents deployment as well as testing of the latter devices requires compliant real-size models of the human aortic root. The casting methods utilized up to now are multi-step, time consuming and complicated. We pursued a goal of building a flexible 3D model in a single-step procedure. We created a precise 3D CAD model of a human aortic root using previously published anatomical and geometrical data and printed it using a novel rapid prototyping system developed by the Fab@Home project. As a material for 3D fabrication we used common house-hold silicone and afterwards dip-coated several models with dispersion silicone one or two times. To assess the production precision we compared the size of the final product with the CAD model. Compliance of the models was measured and compared with native porcine aortic root. Total fabrication time was 3 h and 20 min. Dip-coating one or two times with dispersion silicone if applied took one or two extra days, respectively. The error in dimensions of non-coated aortic root model compared to the CAD design was <3.0% along X, Y-axes and 4.1% along Z-axis. Compliance of a non-coated model as judged by the changes of radius values in the radial direction by 16.39% is significantly different (P<0.001) from native aortic tissue--23.54% at the pressure of 80-100 mmHg. Rapid prototyping of compliant, life-size anatomical models with the Fab@Home 3D printer is feasible--it is very quick compared to previous casting methods.

A new species of Homalocerus Schoenherr from the Atlantic coast of the State of São Paulo, Brazil (Coleoptera, Belidae, Belinae), with notes on color pattern and on the sclerites of the internal sac

A new species of Homalocerus Schoenherr from the Atlantic coast of the State of São Paulo, Brazil (Coleoptera, Belidae, Belinae), with notes on color pattern and on the sclerites of the internal sac. Homalocerus bimaculatus sp. nov. (type locality: Brazil, São Paulo) is described and illustrated, and comments on the sclerites of the internal sac of aedeagus and on color pattern are provided. The new species is compared to other similar species of the genus, being distinguished by having three clusters of carmine pubescence on pronotum and two lateral whitish oval spots located slightly before the middle of each elytron. Six species of Homalocerus, including the new one, are known from the State of São Paulo. The previously published identification key for species of Homalocerus is updated to include H. bimaculatus.