Chloroplast DNA analysis in Tunisian fig cultivars (Ficus carica L.): Sequence variations of the trnL-trnF intergenic spacer

We investigated the nucleotide variation of a non-coding, chloroplast DNA (cpDNA) region to infer relationships among Tunisian fig cultivars. In this study, we examine the level of genetic diversity and its distribution using sequences of the trnL and trnF genes intergenic spacer. The non-coding region displays 28 substitution sites. Insertions and deletions involving 6 sites were found. By using the Kimura-2 method, nucleotide sequences have been aligned using the MEGA program to calculate pairwise divergence of trnL-trnF spacer sequences between cultivars. The size of this non-coding region varied from 430 to 464 bases. The relatively high A + T values (63.7–64.4%) of trnL-trnF intergenic spacer in Ficus carica may explain the high proportion of the identified transversions (t_i/t_v = 0.9). These results suggest the occurrence of nucleotide diversity with a large variation level of chloroplast non-coding region. The analysed data illustrate a considerable level of variability in the genetic pool of the local germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical correspondence. Fourteen haplotypes were detected among 20 individuals examined, yielding a haplotype diversity of 0.983 and a high level of nucleotide diversity (0.0100). The observed variation pattern of plastid DNA provides evidence that the fig germplasm has been undergoing rapid expansion. Neutrality tests rejected the neutrality assumption in the total sample. The cytoplasm variability indicates a narrow genetic base in the cultivated common fig. Despite the high level value of the apparent diversity, we may conclude that fig chloroplast genome provides a new conceptual and practical opportunity to evaluate genetic diversity and to identify local cultivars, making it a valuable source to include into potential breeding programs.     

Antimicrobial and antioxidant activities of methanol extracts of <Emphasis Type="Italic">Evax pygmaea</Emphasis> (Asteraceae) growing wild in Tunisia

Antimicrobial activity of methanolic extracts obtained from the aerial parts of Evax pygmaea was tested against five bacteria and two strains of phytopathogenic fungi using the agar diffusion and broth microdilution methods. Antioxidant properties were evaluated through the ability of the different fractions to scavenge the stable ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals. The TEAC (Trolox Equivalent Antioxidant Capacity) and IC₅₀ values of the fractions were calculated and compared. The experimental data indicated that all fractions exhibit moderate to appreciable antibacterial activities against all Gram-positive cocci and Gram-negative rods except Pseudomonas aeruginosa, but no antifungal activity was observed. Ethyl acetate and methanol fractions were found to cause significant free radical-scavenging effects in both assays. These results may suggest that E. pygmaea could be used as a natural preservative ingredient in the food and/or pharmaceutical industries.     

In vitro selection of external guide sequences for directing RNase P-mediated inhibition of viral gene expression

External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNA in vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application.     

Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate

RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3′ immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5′ immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.     

14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes

The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin alpha6, integrin beta4, or bullous pemphigoid antigen-1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14-3-3 sigma, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3sigma GST pull-down screening assay and showed that sigma (sigma) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3sigma revealed that only intact 14-3-3sigma molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 sigma is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 sigma interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3sigma isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.     

Profiles of the Essential Oils and Headspace Analysis of Volatiles from Mandragora autumnalis Growing Wild in Tunisia

Mandragora autumnalis Bertol . (Solanaceae family), synonym of M. officinalis Mill ., occurs in North Africa and grows natively in Northern and Central Tunisia, in humid to sub‐arid climates. The ripe fruits of mandrake are odiferous with a particular, indescribable, specific odor, shared, to a lesser extent, by the leaves and roots. We carried out an investigation of the essential oils (EOs) and of the aromatic volatiles emitted by fresh leaves, roots and ripe fruits of M. autumnalis growing wild in Central Tunisia. The EOs were obtained from freshly collected plant material by hydrodistillation, while the volatile emissions from the powdered M. autumnalis tissues were sampled by headspace solid phase microextraction (HS‐SPME); both types of samples were analyzed by gas chromatography‐mass spectrometry (GC/MS). Fifty‐one compounds representing 96.2–98.6 % of the total oil compositions were identified in the three tissues and belonged to different chemical classes specifically in 16 esters, 12 alcohols, 12 hydrocarbons, 6 ketones, 3 aldehydes and 3 acids. The main constituents were pentadecanoic acid (34.2 %) and hexadecanol (26.3 %). A total of 78 volatile compounds emanating from M. autumnalis tissues, representing 94.1–96.4 % of the total volatile compositions, were identified: 22 esters, 11 alcohols, 9 aldehydes, 14 ketones, 7 nitrogen, 10 hydrocarbons, 2 lactones, 1 sulfur and 2 ethers. Ethyl hexanoate (12.3 %) and 1,3‐butanediol (12.3 %) were at the highest relative percentages. This study characterizes and distinguishes M. autumnalis from Tunisia and attributes the compounds responsible for the intoxicating and particular odor of fruits. Chemosystematic of Mandragora autumnalis based on the identification of essential oils and headspace volatiles of each of its organ can be used to characterize this species according to its geographic distribution.     

The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.     

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

Potential role of NK cells in future anti-tumor immunotherapies

Natural Killer (NK) cells are no longer considered as relatively unimportant bystander cells having the capacity to kill certain tumor and virus-infected cells in a mysterious way. During the last decade a significant progress has been made in understanding biology of NK cells in particular their mechanisms of recognition and killing of target cells. This progress has led to novel knowledge-based clinical applications of NK cells as immunotherapeutic tools in various disease settings, especially in bone marrow transplantation for leukemia patients. The potential of NK cell therapy for eradicating solid tumors has not been fully exploited. In this mini-review, we examine the rationale behind these therapies and discuss the problems confronting researchers in their usage as therapeutic agents.     

Production of Hexenol in a Two-Enzyme System: Kinetic Study and Modelling

In a two-enzyme system, successive action of hydroperoxide lyase from mint and yeast alcohol-dehydrogenase catalyses the conversion of hydroperoxy linolenic acid to hexenol. Kinetic behaviour was investigated separately for each enzyme: a lumped model based on the Michaelis-Menten approach shows the fate of the reactants in the system.