Modulation of Adenosine 5′-Monophosphate Adsorption Onto Aqueous Resident Pyrite: Potential Mechanisms for Prebiotic Reactions

The adsorption of adenosine 5-monophosphate (5-AMP) ontopyrite (FeS₂) and its modulation by acetate, an organic precursor of complex metabolic pathways, was studied in aqueousmedia that simulate primitive environments. 5-AMP adsorptionrequires divalent cations, indicating that a cationic bridge mediates its attachment to negatively charged sites of the mineral surface. The isotherm of 5-AMP adsorption exhibits a strong cooperative effect at low nucleotide concentrations inacetate-rich medium, whereas high levels of adsorption were only found at high nucleotide concentrations in a model of primitive seawater (acetate free). The modulating role of acetate is also evidenced in the presence of high dipolar moment molecules: dimethyl sulfoxide (Me₂SO) and dimethylformamide (DMF) strongly inhibit 5-AMP adsorption in acetate-rich media, whereas no effect of DMF was found in artificial seawater. The observation that exogenous divalentcations are not needed for acetate attachment onto FeS₂ reveals that organic acids can interact with the Fe²⁺ atoms in the mineral surface. All considered, the results showthat complex and flexible iron-sulfide/biomonomers interactionscan be modulated by molecules that accumulate in the interfacelayer.     

In vivo analysis reveals different apoptotic pathways in pre‐ and postmigratory cerebellar granule cells of rabbit

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly‐ADP‐ribose polymerase‐1 (PARP‐1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437–452, 2004     

Critical notes regarding the dislocation of Chicanos by the Spanish‐language television industry in the United States

The growth of the Spanish‐speaking Hispanic population in the United States has fostered the creation of an extensive Spanish‐language media industry. In particular, while the Spanish‐language media industry has expanded in correspondence with the growth of the Spanish‐speaking Hispanic population in the United States, Spanish‐language television has done so in order to capitalize on the consumer and commercial value of the population. In doing so, we argue in this article that the Spanish‐language media industry serves to dislocate Chicanos in US society by not promoting their sociocultural and ethnolinguistic identity.     

Opportunistic Pathogens and Elements of the Resistome that Are Common in Bottled Mineral Water Support the Need for Continuous Surveillance

Several differences concerning bacterial species, opportunistic pathogens, elements of the resistome as well as variations concerning the CFU/mL counts were identified in some of the five most marketed bottled mineral water from Araraquara city, São Paulo, Brazil. Two out of five brands tested were confirmed as potential source of opportunistic pathogens, including Mycobacterium gordonae, Ralstonia picketti and Burkholderia cepacia complex (Bcc). A total of one hundred and six isolates were recovered from four of these bottled mineral water brands. Betaproteobacteria was predominant followed by Alphaproteobacteria, Gammaproteobacteria and Firmicutes. Ninety percent of the bacteria isolated demonstrated resistance to seventeen of the nineteen antimicrobials tested. These antimicrobials included eight different classes, including 3rd and 4th generation cephalosporins, carbapenems and fluoroquinolones. Multidrug resistant bacteria were detected for fifty-nine percent of isolates in three water brands at counts up to 10³ CFU/ml. Of major concern, the two bottled mineral water harboring opportunistic pathogens were also source of elements of the resistome that could be directly transferred to humans. All these differences found among brands highlight the need for continuous bacteriological surveillance of bottled mineral water.     

Genetic Determinants for Gestational Diabetes Mellitus and Related Metabolic Traits in Mexican Women

Epidemiological and physiological similarities among Gestational Diabetes Mellitus (GDM) and Type 2 Diabetes (T2D) suggest that both diseases, share a common genetic background. T2D risk variants have been associated to GDM susceptibility. However, the genetic architecture of GDM is not yet completely understood. We analyzed 176 SNPs for 115 loci previously associated to T2D, GDM and body mass index (BMI), as well as a set of 118 Ancestry Informative Markers (AIMs), in 750 pregnant Mexican women. Association with GDM was found for two of the most frequently replicated T2D loci: a TCF7L2 haplotype (CTTC: rs7901695, rs4506565, rs7903146, rs12243326; P=2.16x10^-06; OR=2.95) and a KCNQ1 haplotype (TTT: rs2237892, rs163184, rs2237897; P=1.98x10^-05; OR=0.55). In addition, we found two loci associated to glycemic traits: CENTD2 (60’ OGTT glycemia: rs1552224, P=0.03727) and MTNR1B (HOMA B: rs1387153, P=0.05358). Remarkably, a major susceptibility SLC16A11 locus for T2D in Mexicans was not shown to play a role in GDM risk. The fact that two of the main T2D associated loci also contribute to the risk of developing GDM in Mexicans, confirm that both diseases share a common genetic background. However, lack of association with a Native American contribution T2D risk haplotype, SLC16A11, suggests that other genetic mechanisms may be in play for GDM.     

Singlet oxygen quenching by anthocyanin's flavylium cations

The quenching of singlet molecular oxygen (¹O₂) by the flavylium cation form of six widespread anthocyanin derivatives: cyanidin 3-glucoside (CG), cyanidin 3-rutinoside (CR), cyanidin 3-galactoside (CGL), malvidin (M), malvidin 3-glucoside (MG) and malvidin 3,5-diglucoside (MDG) was studied in 1% HCl methanol solution by time-resolved phosphorescence detection (TRPD) of ¹O₂ and photostationary actinometry using perinaphthenone and methylene blue as sensitizers, respectively. The average value of the total (k₀) and chemical (k_c) quenching rate constants were ～ 4×10⁸ m^?1 s^?1 and 3×10⁶ m^?1 s^?1, respectively, indicating the good performance of the studied anthocyanins as catalytic quenchers of ¹O₂. The quenching efficiency was larger for malvidin than for cyanidin derivatives, probably by the extra electron-donating methoxy group in ring B of the malvidin derivatives; and it was also dependent on the number and type of glycosylated substitution. As observed for other phenolic-like derivatives, the quenching of ¹O₂ by anthocyanins was mediated by a charge-transfer mechanism, which was modulated by the total number of –OR substituents that increases the electron-donating ability of these compounds.     

Mesenchymal stem cell conditioned medium increases glial reactivity and decreases neuronal survival in spinal cord slice cultures

Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell (MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of βIII-tubulin immunoreactive neurites, whilst Ca²⁺ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.     

Investigation of charged polymer influence on green fluorescent protein thermal stability

Methods of stabilization and formulation of proteins are important in both biopharmaceutical and biocatalysis industries. Polymers are often used as modifiers of characteristics of biological macromolecules to improve the biochemical activity and stability of proteins or drug bioavailability. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Relative thermal stability was undertaken by incubation of GFP at varying temperatures and GFP fluorescence was used as a reporter for unfolding. At 80°C, DEAE-dextran did not have any effect on GFP fluorescence, indicating that it does not confer stability.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS–protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM_LPS?>?98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K _GFPuv?<?1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein–LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein–LPS aggregation.