Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.     

CD36 or alphavbeta3 and alphavbeta5 integrins are not essential for MHC class I cross-presentation of cell-associated antigen by CD8 alpha+ murine dendritic cells

Cross-presentation of cell-associated Ag is thought to involve receptor-mediated uptake of apoptotic cells by dendritic cells (DC), and studies with human DC strongly implicate the endocytic receptor CD36 and the integrins alpha(v)beta(3) and/or alpha(v)beta(5) in this process. In the mouse, cross-presentation was recently shown to be a function of CD8alpha(+) DC. Here we report that CD36 is expressed on CD8alpha(+), but not on CD8alpha(-), DC. To address the role of CD36 in cross-presentation we compared CD36(-/-) and CD36(+/+) H-2(b) DC for their ability to stimulate naive OT-1 T cells specific for OVA plus H-2K(b) in the presence of OVA-loaded MHC-mismatched splenocytes as a source of cell-associated Ag for cross-presentation. Surprisingly, no difference was seen between CD36(-/-) and CD36(+/+) CD8alpha(+) DC in their ability to cross-present cell-associated OVA or to capture OVA-bearing cells. Furthermore, the proliferation of CFSE-labeled OT-1 cells in response to OVA cross-presentation in vivo was normal in CD36(-/-) bone marrow chimeras, also arguing against a necessary role for CD36 in cross-presentation by DC or other APC. DC doubly deficient for beta(3) and beta(5) integrins were similarly unimpaired in their ability to cross-present OVA-bearing cells in vitro. These data demonstrate that in the mouse, receptors other than CD36 or beta(3) and beta(5) integrins can support the specialized cross-presenting function of CD8alpha(+) DC.     

Coupling between the BLUF and EAL domains in the blue light-regulated phosphodiesterase BlrP1

The first biochemical and structural characterization of the full-length active photoreceptor BlrP1 from Klebsiella pneumoniae was recently reported by Barends et al. [Nature 459:1015–1018, (2009)]. The light-regulated catalytic function of its C-terminal c-di-guanosine monophosphate phosphodiesterase, the EAL (Glu-Ala-Leu) domain, is activated by the N-terminal sensor of blue light using the flavin adenine dinucleotide (BLUF) domain. We performed molecular dynamics simulations on the dimeric BlrP1 protein in order to examine the coupling regions that are presumably involved in transmitting light-induced structural changes which occur in the BLUF domain to the EAL domain. According to the results of simulations and an analysis of the hydrogen bonding between the respective polypeptide chains, the region containing the site on the α3α4 loop of BLUF is responsible for communication between the photosensing and catalytic domains in the dimeric BlrP1 protein.     

Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.     

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.     

Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.     

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.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.