Evaluation of the anti-inflammatory and antinociceptive properties of p-cymene in mice

We attempted to identify the antinociceptive and anti-inflammatory actions of the monoterpene p-cymene. Firstly, behavioural screening was carried out to verify the influence of p-cymene [25, 50, and 100 mg/kg intraperitoneal (i.p.)] on the central nervous system (CNS) activity. The antinociceptive activity of p-cymene was evaluated by the acetic acid-induced writhing response, formalin, and hot-plate test, respectively. The leukocyte migration induced by injection of carrageenan was used to assess the anti-inflammatory activity. p-Cymene showed depressant activity on CNS after 4 h of treatment and also a possible action on the autonomous nervous system (ANS), mainly at the dose of 100 mg/kg (i.p.). It was found that p-cymene (50 and 100 mg/kg, i.p.) significantly (p < 0.05) reduced the writhing responses induced by acetic acid. p-Cymene also decreased the licking time in the first and second phase, respectively, of the formalin test. The results of the hot-plate test showed that all doses of p-cymene increased significantly the latency time of the response to the thermal stimulus in both licking and jumping parameters. In addition, there was a significantly (p < 0.05) decreased leukocyte migration at all doses of p-cymene. The experimental data demonstrate that p-cymene possesses antinociceptive and anti-inflammatory activities.     

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of São Paulo, Brazil

SUMMARY The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the S?o Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.     

Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.Starch is the most important carbohydrate used for food and feed purposes and represents the major resource for our diet (Smith, 2008). The total yield of starch in rice (Oryza sativa), corn (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum) exceeds 10⁹ tons per year (Kossmann and Lloyd, 2000; Slattery et al., 2000). In addition to its use in a nonprocessed form, extracted starch is processed in many different ways, for instance as a high-Fru syrup, as a food additive, or for various technical purposes. As a result of this considerable importance, increasing the starch content of plant tissues has been a major goal for many years, with both classical breeding and biotechnological approaches being taken extensively over the last few decades (Martin and Smith, 1995; Regierer et al., 2002).The pathway by which carbon is converted from Suc to starch in the potato tuber is well established (Kruger, 1997; Fernie et al., 2002; Geigenberger et al., 2004; Geigenberger, 2011). Imported Suc is cleaved in the cytosol by Suc synthase, resulting in the formation of UDP-Glc and Fru; the UDP-Glc is subsequently converted to Glc-1-P by UDP-Glc pyrophosphorylase. The second product of the Suc synthase reaction, Fru, is efficiently phosphorylated to Fru-6-P by fructokinase (Renz et al., 1993; Davies et al., 2005). Fru-6-P is freely converted to Glc-6-P, in which form it normally enters the amyloplast (Kammerer et al., 1998; Tauberger et al., 2000; Zhang et al., 2008), and once in the plastid, it is converted to starch via the concerted action of plastidial phosphoglucomutase, ADP-Glc pyrophosphorylase (AGPase), and the various isoforms of starch synthase (Martin and Smith, 1995; Geigenberger, 2011). Of these reactions, although some of the control of starch synthesis resides in the plastidial phosphoglucomutase reaction (Fernie et al., 2001b), the AGPase reaction harbors the highest proportion of control within the linear pathway (Sweetlove et al., 1999; Geigenberger et al., 1999, 2004). In addition, considerable control resides in both the Glc-6-P phosphate antiporter (Zhang et al., 2008) and the amyloplastidial adenylate transporter (Tjaden et al., 1998; Zhang et al., 2008) as well as in reactions external to the pathways, such as the amyloplastidial adenylate kinase (Regierer et al., 2002), cytosolic UMP synthase (Geigenberger et al., 2005), and mitochondrial NAD-malic enzyme (Jenner et al., 2001).As part of our ongoing study of the constituent enzymes of the tricarboxylic acid (TCA) cycle, we made an initially surprising observation that increasing or decreasing the content of malate via a fruit-specific expression of antisense constructs targeted against the mitochondrial malate dehydrogenase or fumarase, respectively, resulted in opposing changes in the levels of starch (Centeno et al., 2011). We were able to demonstrate that these plants were characterized by an altered cellular redox balance and that this led to changes in the activation state of the AGPase reaction. Given that starch only accumulates transiently in tomato (Solanum lycopersicum; Beckles et al., 2001) as a consequence of this activation, the fruits were characterized by altered sugar content at ripening, a fact that dramatically altered their postharvest characteristics (Centeno et al., 2011). Here, we chose to express the antisense fumarase construct in potato in order to ascertain the effect of the manipulation in an organ that linearly accumulates starch across its development. The results obtained are compared and contrasted with those of the tomato fruit and within the context of current models of subcellular redox regulation.     

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.     

Regional inequalities in cancer care persist in Italy and can influence survival

BackgroundPopulation-based cancer registry studies of care patterns can help elucidate reasons for the marked geographic variation in cancer survival across Italy. The article provides a snapshot of the care delivered to cancer patients in Italy.MethodsRandom samples of adult patients with skin melanoma, breast, colon and non-small cell lung cancers diagnosed in 2003–2005 were selected from 14 Italian cancer registries. Logistic models estimated odds of receiving standard care (conservative surgery plus radiotherapy for early breast cancer; surgery plus chemotherapy for Dukes C colon cancer; surgery for lung cancer; sentinel node biopsy for >1 mm melanoma, vs. other treatment) in each registry compared to the entire sample (reference).ResultsStage at diagnosis for breast, colon and melanoma was earlier in north/central than southern registries. Odds of receiving standard care were lower than reference in Sassari (0.68, 95%CI 0.51–0.90) and Napoli (0.48, 95%CI 0.35–0.67) for breast cancer; did not differ across registries for Dukes C colon cancer; were higher in Romagna (3.77, 95%CI 1.67–8.50) and lower in Biella (0.38, 95%CI 0.18–0.82) for lung cancer; and were higher in Reggio Emilia (2.37, 95%CI 1.12–5.02) and lower in Ragusa (0.27, 95%CI 0.14–0.54) for melanoma.ConclusionsNotwithstanding limitations due to variations in the availability of clinical information and differences in stage distribution between north/central and southern registries, our study shows that important disparities in cancer care persist across Italy. Thus the public health priority of reducing cancer survival disparities will not be achieved in the immediate future.     

Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

Hypomethylation of cytosine residues in cold-sensitive regions of Cestrum strigilatum (Solanaceae)

Heterochromatin comprises a fraction of the genome usually with highly repeated DNA sequences and lacks of functional genes. This region can be revealed by using Giemsa C-banding, fluorochrome staining and cytomolecular tools. Some plant species are of particular interest through having a special type of heterochromatin denominated the cold-sensitive region (CSR). Independent of other chromosomal regions, when biological materials are subjected to low temperatures (about 0 °C), CSRs appear slightly stained and decondensed. In this study, we used Cestrum strigilatum (Solanaceae) to understand some aspects of CSR condensation associated with cytosine methylation levels, and to compare the behavior of different heterochromatin types of this species, when subjected to low temperatures.     

Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde-activated chitosan

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.     

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.