Proteolytic modification of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-512F dextransucrase

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209–Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.Martha Argüello-Morales and Mónica Sánchez-González equally contributed to this work.     

Conservation implications of genetic structure in the narrowest endemic quillwort from the Eastern Amazon

The quillwort Isoëtes cangae is a critically endangered species occurring in a single lake in Serra dos Carajás, Eastern Amazon. Low genetic diversity and small effective population sizes (N _e) are expected for narrow endemic species (NES). Conservation biology studies centered in a single species show some limitations, but they are still useful considering the limited time and resources available for protection of species at risk of extinction. Here, we evaluated the genetic diversity, population structure, N _e, and minimum viable population (MVP) of I. cangae to provide information for effective conservation programs. Our analyses were based on 55 individuals collected from the Amendoim Lake and 35,638 neutral SNPs. Our results indicated a single panmictic population, moderate levels of genetic diversity, and N _e in the order of thousands, contrasting the expected for NES. Negative F_IS values were also found, suggesting that I. cangae is not under risk of inbreeding depression. Our findings imply that I. cangae contains enough genetic diversity to ensure evolutionary potential and that all individuals should be treated as one demographic unit. These results provide essential information to optimize ex situ conservation efforts and genetic diversity monitoring, which are currently applied to guide I. cangae conservation plans.     

Endogamy costs and reproductive biology of <Emphasis Type="Italic">Laelia autumnalis</Emphasis>, an endemic orchid of Mexico

It has been hypothesized that the fitness of selfed progeny must be lowered by inbreeding depression. Most research into the breeding systems of orchids shows a similar fruit set from outcrossing and self-pollination, but few studies have measured seed production and viability. In five populations of Laelia autumnalis, in central México, we studied the species reproductive system and the early costs of endogamy. We performed spontaneous self-pollination, apomixis, assisted self-pollination, cross-pollination, and exogamous pollination (using pollen from a different population) treatments of bagged flowers and measured fruit set, seed production, and germination. No fruits were produced in the apomixis and spontaneous self-pollination treatments. Reproductive success from self-pollination was lower than that from cross- and exogamous pollination and no difference was found between the latter two treatments. The ratio between self-pollination and cross-pollination success in different traits ranged from 0.80?±?0.18 (fruit set) to 0.22?±?0.13 (seeds with embryo). The latter value suggests a high endogamy costs in the production of viable seeds. The concatenated success of the different traits studied showed that the relative fitness of self-pollination was 63% lower than with cross-pollination. Laelia autumnalis is a self-compatible non-autogamous species in which the cost of endogamy occurs at seed production and cannot be compensated for at other early stages. This also occurs in other orchid species and is likely to be a general pattern.     

The Cratylia mollis Seed Lectin Induces Membrane Permeability Transition in Isolated Rat Liver Mitochondria and a Cyclosporine A‐Insensitive Permeability Transition in Trypanosoma cruzi Mitochondria

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca²⁺ and mitochondrial dysfunction due to matrix Ca²⁺ overload. In order to investigate the mechanism of Ca²⁺‐induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (?Ψ_m) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca²⁺ was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca²⁺ this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ?Ψ_m disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca²⁺ dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA‐insensitive MPT in T. cruzi mitochondria.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Male age and strain affect ejaculate quality in the Mexican fruit fly

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass‐reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass‐reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass‐reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass‐reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.     

Spatial and temporal variation in breeding parameters of two south-temperate populations of House Wrens

Studies of variation in breeding parameters are often based on temporal analyses of a single population. However, to differentiate between the effects of regional and local factors, neighboring populations with limited interpopulational dispersal need to be compared. We studied two nearby (< 5 km apart) populations of House Wrens (Troglodytes aedon bonariae) at two ranches (Los Zorzales, 10 years; La Esperanza, 13 years) in south-temperate Argentina to assess the possible effects of regional and local factors on breeding phenology. For each breeding season, we recorded laying dates, clutch sizes, and length of the breeding season, and estimated the reproductive synchrony of first and second breeding attempts. We examined how these breeding parameters were affected by weather, population density, and rates of nest failure. With favorable temperatures during the pre-reproductive period (September–October), wrens in both populations initiated first breeding attempts earlier. However, ordinal laying dates were also affected by local factors, with wrens at Los Zorzales initiating breeding attempts earlier than those at La Esperanza. We found a spatial correlation in clutch sizes between populations for the 2007–2012 breeding seasons, but clutch sizes of first and second nesting attempts showed low variability. Reproductive synchrony of first nesting attempts varied among years, suggesting an effect of regional factors. However, we detected no synchronization between populations and were unable to identify environmental variables that explained the temporal variation. Ordinal laying dates of second clutches were strongly correlated with the ordinal laying dates of first clutches. We also found that the length of breeding seasons was longer when daily nest mortality rates were lower. Although environmental factors seemed to affect the decision of when to start breeding, pairs with successful first nesting attempts were more likely to initiate second nests, thus affecting the length of the breeding season. The spatial variation and temporal variation of the breeding parameters of House Wrens in our study provide evidence of marked plasticity in their breeding decisions and allowed us to identify local and regional environmental factors related to this variation.     

Carbon Monoxide-releasing Molecule-3 (CORM-3; Ru(CO)3Cl(Glycinate)) as a Tool to Study the Concerted Effects of Carbon Monoxide and Nitric Oxide on Bacterial Flavohemoglobin Hmp: APPLICATIONS AND PITFALLS*

CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O₂ to the ion nitrate (NO₃⁻). The protoheme of Hmp binds not only O₂ and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)₃Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo.