In vitro anthelmintic effect of Melia azedarach L. and Trichilia claussenii C. against sheep gastrointestinal nematodes

The control of parasitic diseases in small ruminants is mainly done with the use of synthetic anthelmintics. However, incorrect and indiscriminate use of these products has caused the emergence of parasite resistance. Plants with anthelmintic activity are used in folk veterinary medicine, but it is necessary to investigate and scientifically validate low-cost phytotherapeutic alternatives for future use to control gastrointestinal nematodes in small ruminants by family farmers. Thus, the aim of this study was to evaluate the in vitro anthelmintic effect of plant extracts from Melia azedarach and Trichilia claussenii by the egg hatch test (EHT) and larval development test (LDT) against sheep gastrointestinal nematodes. The hexane extract of M. azedarach fruits was extracted through cold percolation and the methanol extract of T. claussenii leaves was obtained by extraction at room temperature in solvents in order of increasing polarity. The efficacy results were analyzed using the Probit program of SAS. The M. azedarach extract showed a LC(50) of 572.2 μg/mL and LC(99) of 1137.8 μg/mL in the EHT, and LC(50) of 0.7 μg/mL and LC(99) of 60.8 μg/mL in the LDT. In turn, the T. claussenii extract presented a LC(50) of 263.8 μg/mL and LC(99) of 522.5 μg/mL in the EHT and LC(50) of 1.1 μg/mL and LC(99) of 26.4 μg/mL in the LDT. Comparing the extracts of the species from the Meliaceae family, T. claussenii showed greater anti-parasite potential in vitro than M. azedarach. However, studies on the isolated compounds, toxicity and administration forms to animals are also needed to validate low-cost alternative herbal remedies for use to control gastrointestinal nematodes by family farmers.     

Scale formation as related to length of young-of-the-year Ide Idus idus and roach Rutilus rutilus

The scale formation in relation to length of young-of-the-year ide Idus idus (L.) and roach Rutilus rutilus (L.) is described for fish from River Kävlingeän, South Sweden. The appearance of the first scale is noted as well as the scale pattern formation.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1) during osmotic cell shrinkage

The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.     

Binding of [3H]ouabain to split frog skin: the role of the Na,K-ATPase in the generation of short circuit current

The binding of [3H]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (Isc) was concurrently monitored. Both ouabain binding and Isc inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Isc inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Isc. Upon separation of the skins into two groups based upon initial Isc(Isci)--high, greater than 20 microamperemeter/cm2 and low, less than 10 microamperemeter/cm2, we observed two distinct populations. The high Isci skins bound very little ouabain before inhibition of Isc whereas low Isci skins bound one-half of the total number of sites before exhibiting any inhibition of Isc. These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Isc, and (b) at low Isc, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Isc relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: (a) increased intracellular K/Na concentrations, whether achieved through the addition of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and (b) ouabain binding, either with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number of ouabain molecules bound to the frog skins was not correlated with their initial Isc values, indicating that the spontaneous skin-to-skin variation in Isc was not related to the number of functional pump sites but, rather, to their turnover rate.     

Angiostrongylus cantonensis infection in molluscs in the municipality of S?o Gon?alo,a metropolitan area of Rio de Janeiro,Brazil: role of the invasive species Achatina fulica in parasite transmission dynamics

The aim of this study was to analyse the infection dynamics ofAngiostrongylus cantonensis in its possible intermediate hosts over two years in an urban area in the state of Rio de Janeiro where the presence ofA. cantonensis had been previously recorded in molluscs. Four of the seven mollusc species found in the study were exotic.Bradybaena similaris was the most abundant, followed byAchatina fulica, Streptaxis sp., Subulina octona, Bulimulus tenuissimus, Sarasinula linguaeformis and Leptinaria unilamellata. Only A. fulica and B. similaris were parasitised by A. cantonensis and both presented co-infection with other helminths. The prevalence of A. cantonensisin A. fulica was more than 50% throughout the study. There was an inverse correlation between the population size ofA. fulica and the prevalence of A. cantonensis and abundance of the latter was negatively related to rainfall. The overall prevalence of A. cantonensis in B. similariswas 24.6%. A. fulica was the most important intermediary host of A. cantonensis in the studied area andB. similaris was secondary in importance for A. cantonensis transmission dynamics.     

NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity

The interaction of the ubiquitous Na(+)/H(+) exchanger, NHE1, with its commonly used inhibitors, amiloride- and benzoylguanidine (Hoechst type inhibitor (HOE))-type compounds, is incompletely understood. We previously cloned NHE1 from Amphiuma tridactylum (AtNHE1) and Pleuronectes americanus (PaNHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1 regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10-11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity. The converse mutant (TM10-11 of AtNHE1 replaced with those of PaNHE1) exhibited even higher amiloride and EIPA sensitivity and was also HOE-sensitive. Replacing an LFFFY motif in TM region 4 of PaNHE1 with the corresponding residues of hNHE1 (VFFLF) or AtNHE1 (TFFLF) greatly increased sensitivity to both amiloride- and HOE-type compounds, despite the fact that AtNHE1 is HOE694-insensitive. Gain of amiloride sensitivity appeared to correlate with increased Na(+)/H(+) exchange rates. It is concluded that regions within TM4 and TM10-11 contribute to amiloride and HOE sensitivity, with both regions imparting partial inhibitor sensitivity to NHE1.     

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2^LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.     

Morphological identification of ticks (Acari: Ixodidae) infesting donkeys (Equus asinus) in Maputo Province,Mozambique

Donkeys (Equus asinus) are rustic animals, but in Africa’s poorest regions, they can present multiple health problems, including tick infestation. The study’s objective was to determine the species composition of ticks that infest donkeys in Maputo Province (Mozambique). Ticks were collected in five conveniently selected southern districts of Maputo Province (Moamba, Matutuíne, Marracuene, Boane, and Matola) and were identified to species level using a stereoscopic microscope with the aid of dichotomous identification keys. In total, 500 ticks were collected from all 88 selected donkeys. Three genera of ticks were identified, namely Rhipicephalus (97.2%; 486/500), Amblyomma (2.2%; 11/500), and Hyalomma (0.6%; 3/500). Seven species were identified, of which Rhipicephalus evertsi evertsi with 50.4% (252/500) was the most prevalent, followed by Rhipicephalus appendiculatus (27.4%; 137/500), Rhipicephalus turanicus (11.6; 10/500), Rhipicephalus (boophilus) microplus (6.8; 20/500), Amblyomma hebraeum (2.2%; 11/500), Rhipicephalus sanguineus (1%; 5/500) and Hyalomma truncatum (0.6%; 3/500). Rhipicephalus evertsi evertsi occurred in all locations, whereas Hyalomma truncatum occurred only in the Boane district. Males were the most prevalent (67.2%; 336/500). The study revealed that donkeys in Maputo Province were infested with seven tick species of which R. evertsi evertsi was the main species.