Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays

Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.     

Antiplasmodial activity study of angiotensin II via Ala scan analogs

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid‐phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide‐capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile⁵ or the His⁶ residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala⁶]‐AII (79%), and [Ala⁵]‐AII (75%). Analogs [Ala³]‐AII, [Ala¹]‐AII, and AII‐NH₂ showed antiplasmodial activity around 65%, whereas the activity of the [Ala⁸]‐AII, [Ala⁷]‐AII, [Ala⁴]‐AII, and [Ala²]‐AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β‐fold conformation in different solutions. All AII analogs, except [Ala⁴]‐AII and [Ala⁸]‐AII, show contractile responses and interact with the AT₁ receptor, [Ala⁵]‐AII and [Ala⁶]‐AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.     

The Bacterium Endosymbiont of Crithidia deanei Undergoes Coordinated Division with the Host Cell Nucleus

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.     

Phylogeography and diversification of an Amazonian understorey hummingbird: paraphyly and evidence for widespread cryptic speciation in the Plio‐Pleistocene

Straight‐billed Hermit Phaethornis bourcieri inhabits the understorey of upland terra firme forest throughout most of the Amazon basin. Currently, two allopatric taxa regarded as subspecies are recognized: P. b. bourcieri and P. b. major. However, the validity, interspecific limits and evolutionary history of these taxa are not yet fully elucidated. We use molecular characters to propose a phylogenetic hypothesis for populations and taxa grouped under Phaethornis bourcieri. Our results showed that P. bourcieri is part of the ‘Ametrornis’ clade, along with Phaethornis philippii and Phaethornis koepckeae, and that the subspecies major is more closely related to the latter two species than to populations grouped under nominate bourcieri. Our phylogenetic hypotheses recovered three main reciprocally monophyletic clades under nominate bourcieri separated by the lower Negro River and the Branco River or the Branco–Negro interfluve (clades B and C) and the upper Amazon (Solimões) or lower Marañon/Ucayali Rivers (clades C and D). Based on multi‐locus phylogeographic and population genetics approaches, we show that P. b. major is best treated as a separate species, and that P. b. bourcieri probably includes more than one evolutionary species, whose limits remain uncertain. The diversification of the ‘Ametrornis’ clade (P. bourcieri, P. philippii and P. koepckeae) is centred in the Amazon and appears to be closely linked to the formation of the modern Amazon drainage during the Plio‐Pleistocene.     

Responses of a Specialized Insectivorous Mammal (Myrmecophaga tridactyla) to Variation in Ambient Temperature1

This study reports the effect of daily mean ambient temperature on daily activity and habitat use by the giant anteater (Myrmecophaga tridactyla) in the Pantanal wetlands of Brazil. We equipped 11 giant anteaters with a global position system collar programmed to acquire a location every 10 min for 15 d. A temperature data logger left in the meteorological station at the study site registered data on ambient temperature. Giant anteaters were mainly active in open fields except during the coldest days, when they sought protection inside forests. Giant anteaters also used open habitats for resting but during the hottest or chilliest days they sheltered in forests. In the Pantanal, where temperatures are usually high, giant anteaters were active all night long, but as daily average ambient temperature decreased anteaters began and finished activity progressively earlier and reduced total activity. As a consequence, time spent active at daylight increased progressively and time spent active during the night decreased progressively, probably to allow the anteaters to expose themselves to solar radiation and to avoid heat loss during the night.     

Available lysine content in human milk: stability during manipulation prior to ingestion

Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4 degrees C for 48 hours, and frozen for 15 days at -20 degrees C); (b) Thermal treatment under conditions of low (Holder)(63 degrees C/30 minutes) and high pasteurization (75 degrees C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. CONCLUSIONS: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75 degrees C, 15 seconds) than habitual pasteurization (63 degrees C, 30 minutes).     

Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice

Summary Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.