Evidence for aluminum-binding erythropoietin by size-exclusion chromatography coupled to electrothermal absorption atomic spectrometry

Erythropoietin (EPO) is a glycoprotein that stimulates erythropoiesis and is clinically used for treating anemia during chronic renal failure and for anemia in preterm infants. EPO formulations usually have elevated rates of contamination due to aluminum (Al), which is toxic to both types of patients. Size-exclusion chromatography (SEC) coupled with graphite furnace atomic absorption spectrometry (GF AAS) was employed to separate proteins and to quantify the amount of aluminum present in the elution volume corresponding to EPO and, therefore, to evaluate possible binding. Because EPO formulations contain human serum albumin (HSA), a chromatographic method was optimized for the separation of these proteins. Subsequent to the chromatographic separation, 1-mL fractions of the column effluent were collected, and the Al content in these aliquots was measured by GF AAS. EPO and HSA samples were incubated with Al for 4 h at 4 °C and 37 °C as well as for 16 h at 4 °C and 37 °C. Afterwards, they were injected into the chromatographic system. These samples were also submitted to ultrafiltration (10 and 50 kDa membranes), and Al was measured in the ultrafiltrates. The results showed that Al was present in the eluent volume corresponding to the EPO peak but not in the HSA peak in the chromatograms. Temperature strengthened the interaction because the Al present in the EPO fraction was 3 times higher at 37 °C compared to 4 °C. Thirty-eight percent of the Al present in a 2.4 μg/mL EPO standard solution, and approximately 50% of the Al in formulation samples containing approximately 11 μg/mL EPO and either citrate or phosphate, were non-ultrafiltrable, which suggests that EPO is an effective Al acceptor in vitro.     

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.     

Demonstration of reverse fatty acid transport from rat cardiomyocytes

Fatty acids flow from adipocytes to nonadipose tissues during fasting and exercise and normally are fully oxidized. To determine if nonadipose tissues can export unoxidized FA when FA influx exceeds oxidation, neonatal cardiomyocytes were cultured in 1 microCi (14)C-palmitate in the presence of etomoxir to block oxidation. The cells took up and stored 25% of the radioactivity as (14)C-triacylglycerol in 12 h, but 4.5% of the label was released in 3 h and comigrated with (14)C-palmitate. Both uptake and release of radioactivity were increased by insulin and reduced by the nonspecific inhibitors of FA transporters phloretin and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Perfused hearts from etomoxir-treated lean rats released 221 +/- 59 nmol/10 min of FA. Hearts from high-fat-fed lean rats released 366 +/- 172 nmol/10 min (P < 0.05). Hearts from obese rats released 744 +/- 260 and 1,578 +/- 630 nmol/10 min at 8 and 12 weeks of age, respectively. Perfusion with insulin increased FA release by 32%. In vitro and ex vivo findings suggest that nonadipose tissues such as myocardium can export FA when the unoxidized lipid content is excessive.     

Natural cavity restoration as an alternative to nest box supplementation

Nest box supplementation is widely used to increase nest‐site availability for cavity nesting animals but the analysis of its effects on individuals breeding in natural cavities is often neglected. This study offers a novel restoration technique to revert abandonment of natural breeding sites by a secondary cavity avian bird, the European roller (Coracias garrulus), and other ecologically similar species. We found that, after a program of nest box supplementation with ensuing monitoring, rollers gradually abandon nesting in natural and seminatural cavities in favor of nest boxes because the latter are of higher quality. We examine whether reducing the entrance size of natural and seminatural cavities improves their suitability for rollers. A 6‐year program reduced the diameter of the entrance of sandstone cavities and cavities in bridges. This led to a high occupancy (59%) of manipulated nest‐sites. Manipulated sites were most frequently occupied by rollers and little owls (Athene noctua) (31 and 18% of sites, respectively). Manipulation did not affect clutch size or fledgling success. We suggest that nest‐site diversity and nesting in natural cavities should be preserved to reduce nest box dependence. Our study illustrates the value of nest boxes when used alongside restoration of natural breeding sites and provides insights for the management of natural cavities.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Involvement of peripheral TRPV1 in TMJ hyperalgesia induced by ethanol withdrawal

Ethanol withdrawal increases nociception after the injection of formalin into the rat's temporomandibular joint (TMJ). Little is known about the neurological basis for hyperalgesia induced by ethanol withdrawal, but it has been reported that ethanol can potentiate the response of transient receptor potential vanilloid receptor-1 (TRPV1) in superficial tissues. The present study was designed to test the hypothesis that peripheral TRPV1 could be involved on nociceptive behavioral responses induced by the injection of formalin into the TMJ region of rats exposed to chronic ethanol administration and ethanol withdrawal. Behavioral hyperalgesia was verified 12 h after ethanol withdrawal in rats that drank an ethanol solution (6.5%) for 10 days. In another group submitted to the same ethanol regimen, the selective vanilloid receptor antagonist capsazepine (300, 600 or 1200 microg/25 microl) or an equal volume of vehicle were injected into the TMJ regions 30 min before the TMJ formalin test. The local injections of capsazepine reduced the increased nociceptive responses induced by ethanol withdrawal. The effect of capsazepine on rats that did not drink ethanol was not significant. These results indicate that the peripheral TRPV1 can contribute to the hyperalgesia induced by ethanol withdrawal on deep pain conditions.     

The influence of cholesterol on the interaction of HIV gp41 membrane proximal region-derived peptides with lipid bilayers

A small amino acid sequence (LWYIK) inside the HIV-1 gp41 ectodomain membrane proximal region (MPR) is commonly referred to as a cholesterol-binding domain. To further study this unique and peculiar property we have used fluorescence spectroscopy techniques to unravel the membrane interaction properties of three MPR-derived synthetic peptides: the membrane proximal region peptide-complete (MPRP-C) which corresponds to the complete MPR; the membrane proximal region peptide-short (MPRP-S), which corresponds to the last five MPR amino acid residues (the putative cholesterol-binding domain) and the membrane proximal region peptide-intermediate (MPRP-I), which corresponds to the MPRP-C peptide without the MPRP-S sequence. MPRP-C and MPRP-I membrane interaction is largely independent of the membrane phase. Membrane interaction of MPRP-S occurs for fluid phase membranes but not in gel phase membranes or cholesterol-containing bilayers. The gp41 ectodomain MPR may have a very specific function in viral fusion through the concerted and combined action of cholesterol-binding and non-cholesterol-binding domains (i.e. domains corresponding to MPRP-S and MPRP-I, respectively).     

Oocyst ingestion as an important transmission route of Toxoplasma gondii in Brazilian urban children

Toxoplasmosis is a cosmopolitan protozoan infection. Data regarding risk factors for the post-natal acquisition of Toxoplasma gondii infection in childhood are limited. We conducted a serological survey for T. gondii IgG antibodies and associated risk factors in 1,217 children 4-11-yr-old from Salvador, Brazil, using a commercial ELISA kit; antibodies were found in 17.5% of the children. Age (OR = 2.18; 95% CI: 1.50-3.17) and maternal schooling level (OR = 0.62; 95% CI: 0.42-0.92) were negatively associated with infection. A greater number of siblings (OR = 1.53; 95% CI: 1.12-2.09), cat at home (OR = 1.54; 95% CI: 1.06-2.24), house with non-treated piped water (OR = 2.54; 95% CI: 1.22-5.31), and the absence of a flush toilet at home (OR = 1.45; 95% CI: 1.04-2.01) were positively associated with T. gondii infection. Our data suggest that low socioeconomic levels and poor hygiene habits are important factors in favoring T. gondii infection.     

Population growth rate of the depredating Podisus nigrispinus (Heteroptera: Pentatomidae) and of the Tuta absoluta (Leptoptera: gelechiidae) in wintering place

The fertility life table of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) preying either on Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) caterpillars or on alternative prey Tenebrio molitor L. (Coleoptera: Tenebrionidae) under greenhouse conditions (30 +/- 5 degrees C, 61 +/- 23% RH) were studied. The life table was also determined for the pest T. absoluta under the same conditions. The net reproductive rate (Ro) and the intrinsic rate of natural increase (rm) were higher 14.13 and 46.32 times for predators fed on T. molitor prey, however, the generation time (T) was similar between prey. The pest T. absoluta showed Ro and rm higher 2.15 and 32.10 times than those achieved for predators fed on this pest. However, females fed on a suitable prey T. molitor showed higher Ro and rm than those yielded for the pest. The survival curves were similar for P. nigrispinus females fed on both prey and classified as being type II by Weibull analysis. The results suggest that P. nigrispinus is able to maintain its population preying only on T. absoluta caterpillars; however, the life table parameters determined individually for both showed that the pest produces more generations per year and faster population natural growth than the predator.