Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.     

Regulatory phosphorylation of FXYD2 by PKC and cross interactions between FXYD2, plasmalemmal Ca-ATPase and Na,K-ATPase

FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P₂) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca²⁺-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P₂ increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P₂ (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.     

Dynamics of polymerization shed light on the mechanisms that lead to multiple amyloid structures of the prion protein

It is generally accepted that spongiform encephalopathies result from the aggregation into amyloid of a ubiquitous protein, the so-called prion protein. As a consequence, the dynamics of amyloid formation should explain the characteristics of the prion diseases: infectivity as well as sporadic and genetic occurrence, long incubation time, species barriers and strain specificities. The success of this amyloid hypothesis is due to the good qualitative agreement of this hypothesis with the observations. However, a number of difficulties appeared when comparing quantitatively the in vitro experimental results with the theoretical models, suggesting that some differences should hide important discrepancies. We used well defined quantitative models to analyze the experimental results obtained by in vitro polymerization of the recombinant hamster prion protein. Although the dynamics of polymerization resembles a simple nucleus-dependent fibrillogenesis, neither the initial concentration dependence nor off-pathway hypothesis fit with experimental results. Furthermore, seeded polymerization starts after a long time delay suggesting the existence of a specific mechanism that takes place before nucleus formation. On the other hand, polymerization dynamics reveals a highly stochastic mechanism, the origin of which appears to be caused by nucleation heterogeneity. Moreover, the specific structures generated during nucleation are maintained during successive seeding although a clear improvement of the dynamics parameters (polymerization rate and lag time) is observed. We propose that an additional on-pathway reaction takes place before nucleation and it is responsible for the heterogeneity of structures produced during prion protein polymerization in vitro. These amyloid structures behave like prion strains. A model is proposed to explain the genesis of heterogeneity among prion amyloid.     

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

Stability Study of Crude Dextransucrase from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> NRRL B-742

In the present work, the stability of crude dextransucrase from Leuconostoc citreum B-742 was evaluated in synthetic and in cashew apple juice culture broth. Optimum stability conditions for dextransucrase from L. citreum B-742 were different from the reported for its parental industrial strain enzyme (L. mesenteroides B-512F). Crude dextransucrase, from L. citreum B-742, produced using cashew apple juice as substrate, presented higher stability than the crude enzyme produced using synthetic culture medium, showing the same behavior previously reported for dextransucrase from L. mesenteroides B-512F. The crude enzyme presented good stability in cashew apple juice for 48 h at 25°C and pH 6.5.     

Evaluation of the Origin of a Sample of <Emphasis Type="Italic">Sporothrix Schenckii</Emphasis> that Caused Contamination of a Researcher in Southern Brazil

We report a case of a researcher from a laboratory of Mycology in Rio Grande do Sul, Brazil that presented a clinical evidence of sporotrichosis. The researcher had an accident while manipulating the microculture slides of chromoblastomycosis agents and presented a clinical evidence of sporotrichosis. As the laboratory has some cultures of Sporothrix schenckii, it was suggested that it might be a laboratory contamination. In order to test this hypothesis, the genotypic characterization of the samples was performed by means of the random amplified polymorphic DNA (RAPD) analysis method. In addition, we evaluated the in vitro antifungal activity of four antifungal agents against the isolated fungus. The sample obtained from the researcher was not genetically similar to any of the samples kept in the laboratory and showed the minimum inhibitory concentrations of 0.5 μg/mL for itraconazole and ketoconazole, >64 μg/mL for fluconazole and 0.125 μg/mL for terbinafine. It is suggested that the contamination had an environmental origin.     

Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (Decapoda,Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.     

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.