Volatile profiling of Arnicão (Lychnophora salicifolia mart.), a wild medicinal species from Brazilian Cerrado

Abstract

The Cerrado is a diverse Brazilian savanna with more than 12,000 species. Arnicão (Lychnophora salicifolia Mart – Compositae) is an endemic species occurring in central and southeast Brazil, at higher altitudes, in sandstone and quartzite soils. Lychnophora species have been reported for anti-inflammatory, antioxidant and UV protectant effects. Local communities use it to prepare traditional remedies (i.e. ointments or creams). The aim of this work was (1) to characterize the qualitative composition of L. salicifolia volatile fraction; (2) to evaluate the influence of the environmental conditions on the volatile fraction composition, by investigating plants harvested both in areas under stress conditions and protected from human interference; (3) optimizing a non-separative method for routine in-field analyses. Separative (HS-SPME-GC-MS) and non-separative (HS-SPME-MS) approach combined with PCA are proposed to discriminate between plant populations of L. salicifolia from two distinct areas. Forty-eight individuals were randomly collected from four populations. A clear separation between populations from protected and non-protected areas was observed, with an important presence of caryophyllene derivatives in populations from non-protected areas, usually with compounds associated to plant defense. Both analytical approaches gave the same results, confirming HS-SPME a useful approach for rapid in-field analysis of samples of wild populations.     

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-α Using Oriented Peptide Library Screening

Importin-α is the nuclear import receptor that recognizes the classic monopartite and bipartite nuclear localization sequences (cNLSs), which contain one or two clusters of basic amino acids, respectively. Different importin-α paralogs in a single organism are specific for distinct repertoires of cargos. Structural studies revealed that monopartite cNLSs and the C-terminal basic clusters of the bipartite cNLSs bind to the same site on importin-α, termed the major cNLS-binding site. We used an oriented peptide library approach with five degenerate positions to probe the specificity of the major cNLS-binding site in importin-α. We identified the sequences KKKRR, KKKRK, and KKRKK as the optimal sequences for binding to this site for mouse importin-α2, human importin-α1, and human importin-α5, respectively. The crystal structure of mouse importin-α2 with its optimal peptide confirmed the expected binding mode resembling the binding of simian virus 40 large tumor-antigen cNLS. Binding assays confirmed that the peptides containing these sequences bound to the corresponding proteins with low nanomolar affinities. Nuclear import assays showed that the sequences acted as functional cNLSs, with specificity for particular importin-αs. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-α; the results will contribute to understanding of the sequence determinants of cNLSs, and may help identify as yet unidentified cNLSs in novel proteins.     

Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

Pfaffia glomerata (Spreng.) Pedersen is a medicinal species of great interest because it produces the phytoecdysteroid 20-hydroxyecdysone (20E). Generally, because of atypical growing conditions, in vitro propagated plants function less efficiently as autotrophs and have poorly developed morphological structures. This study analyzed the autotrophic potential of P. glomerata propagated in vitro and evaluated the influence that this has on 20E biosynthesis. Physiological and structural parameters of plants subjected to heterotrophic, photomixotrophic and photoautotrophic growth conditions were evaluated. Levels of 20E were measured by HPLC. Plants were acclimatized in a mixture of soil, sand and substrate, in a greenhouse. Conditions that provided higher carbon input led to an increase in plant growth, and the presence of sucrose was critical, in closure systems without a gas permeable membrane, for normal anatomical development of the micropropagated plants. The absence of sucrose increased photosynthesis and conditions that enhanced photoautotrophy induced greater levels of 20E. The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants.     

Does Cry1Ac Bt‐toxin impair development of worker larvae of Africanized honey bee?

The western honey bee (Apis mellifera L.) is a widespread pollinator species. The present study aimed to test if Africanized honey bee larvae are negatively affected by the ingestion of diet contaminated with the Bacillus thunringiensis toxin Cry1Ac, which is expressed in GM cotton plants. The toxin activity was confirmed in bioassays with the velvetbean caterpillar (Anticarsia gemmatalis), a soybean pest species susceptible to Cry1Ac. The honey bee larvae were subjected to ingestion of either pure larval diet (control), diluted larval diet (diluted control) or larval diet diluted in a Cry1Ac solution at a concentration compatible with the maximum possible field exposure. Although diluted diet slightly increased larval mortality, Cry1Ac ingestion did not affect survival, developmental time, and neither adult body mass nor size, indicating that GM plants are unlikely to significantly impair the development of honey bee larvae. The larval‐rearing system reported here was suitable to assess the lethal and sub‐lethal effects of GM expressed toxins against honey bee larvae.     

Mental patients in prisons

Mental conditions usually affect cognitive, emotional and volitional aspects and functions of the personality, which are also functions of interest in law, as they are essential at the time of adjudicating guilt, labeling the accused a criminal, and proffering a sentence. A relationship between mental illness and criminality has, thus, been described and given as one of the reasons for the large number of mental patients in prisons. Whether this relationship is one of causality or one that flows through many other variables is a matter of debate, but there is no debating that prisons have become a de facto part, and an important one, of mental health systems in many countries. This paper deals with the issue of the relationship and provides estimates of prevalence of mental patients in prisons culled from many studies in different countries. It also provides some direction for the management of mental patients as they crowd correctional systems.     

The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Combinatorial regulation modules on GmSBP2 promoter: a distal cis-regulatory domain confines the SBP2 promoter activity to the vascular tissue in vegetative organs

The Glycine max sucrose binding protein (GmSBP2) promoter directs phloem-specific expression of reporter genes in transgenic tobacco. Here, we identified cis-regulatory domains (CRD) that contribute with positive and negative regulation for the tissue-specific pattern of the GmSPB2 promoter. Negative regulatory elements in the distal CRD-A (-2000 to -700) sequences suppressed expression from the GmSBP2 promoter in tissues other than seed tissues and vascular tissues of vegetative organs. Deletion of this region relieved repression resulting in a constitutive promoter highly active in all tissues analyzed. Further deletions from the strong constitutive -700GmSBP2 promoter delimited several intercalating enhancer-like and repressing domains that function in a context-dependent manner. Histochemical examination revealed that the CRD-C (-445 to -367) harbors both negative and positive elements. This region abolished promoter expression in roots and in all tissues of stems except for the inner phloem. In contrast, it restores root meristem expression when fused to the -132pSBP2-GUS construct, which contains root meristem expression-repressing determinants mapped to the 44-bp CRD-G (-136 to -92). Thus, the GmSBP2 promoter is functionally organized into a proximal region with the combinatorial modular configuration of plant promoters and a distal domain, which restricts gene expression to the vascular tissues in vegetative organs.     

Xyloglucan is recognized by carbohydrate-binding modules that interact with beta-glucan chains

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of beta-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a beta-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated beta-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.