Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.     

Normal bone turnover markers in a patient with active Paget's disease of bone: response to treatment with zoledronic acid

The treatment of Paget's disease of bone (PDB) aims at the suppression of abnormal bone turnover; bisphosphonates are currently the treatment of choice. Indications for antiresorptive treatment in symptomatic patients with PDB include bone or joint pain, neurological complications, surgery planned at an active pagetic site and hypercalcaemia from immobilisation. The goals of antiresorptive treatment are clinical improvement and biochemical remission, as assessed by the normalisation of bone turnover markers. Clinical deterioration, especially bone pain, should be considered before deciding to treat patients with late sclerotic (burned-out) PDB. Bone scintigraphy may be of importance in these patients, because it depicts increased osteoblastic activity, when bone markers may not. We present a case of late sclerotic PDB with clinical deterioration but normal bone turnover markers, who experienced significant clinical improvement after treatment with zoledronic acid.     

Τhe Role of a Gibberellin 20-Oxidase Gene in Fruit Development in Pepper (Capsicum annuum)

Fruit shape is a very important fruit quality character frequently affected by grafting in vegetable plants like pepper. It has already been shown that, similar to tomato, fruit shape in pepper is likely controlled by an Ovate-like gene, CaOvate, the down-regulation of which positively affects fruit elongation. To further understand the molecular mechanisms involved in pepper fruit shape control and the changes imposed by grafting, we have amplified, sequenced, and structurally characterized CaGA20ox1, the target gene of CaOvate, from a long fruit and a round fruit shaped cultivar. The results show that CaGA20ox1 has similar genomic organization to the tomato GA20ox1 and encodes a 375-amino acid polypeptide that shares 89% identity with tomato GA20ox1. We then studied CaGA20ox1 expression in different pepper plant parts and in different developmental stages of flower and fruit development. The expression of the gene was quantified by means of relative quantitative PCR in the developmental stage of 10?days after anthesis fruit of both cultivars. The results showed that there is a significant difference in the expression of the CaGA20ox1 between the two cultivars in this specific stage as well as in the expression of CaGA20ox1 after virus-induced gene silencing (VIGS) of CaOvate. Finally, the 5?? upstream sequences of CaGA20ox1 gene of the two cultivars were examined and compared. These results corroborate our previous findings, where VIGS of CaOvate alters CaGA20ox1 expression, leading to more elongated fruit, and also progress further the understanding of the genes involved in fruit shape control in pepper opening the way for understanding the molecular means of grafting effects.     

Biological evaluation of cobalt(II) complexes with non-steroidal anti-inflammatory drug naproxen

Cobalt(II) complexes with the non-steroidal anti-inflammatory drug naproxen in the presence or absence of nitrogen-donor heterocyclic ligands (pyridine, 2,2′-bipyridine or 1,10-phenanthroline) have been synthesized and characterized with physicochemical and spectroscopic techniques. The deprotonated naproxen acts as monodentate ligand coordinated to Co(II) ion through a carboxylato oxygen. The crystal structure of [bis(aqua)bis(naproxenato)bis(pyridine)cobalt(II)], 2 has been determined by X-ray crystallography. The EPR spectrum of complex 2 in frozen solution reveals that it retains its structure. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA and [(2,2′-bipyridine)bis(methanol)bis(naproxenato)cobalt(II)] exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes recorded in DMSO solution and in the presence of CT DNA in 1/2 DMSO/buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that they can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. Naproxen and its cobalt(II) complexes exhibit good binding propensity to human or bovine serum albumin proteins having relatively high binding constant values. The antioxidant activity of the compounds has been evaluated indicating their high scavenging activity against hydroxyl free radicals and superoxide radicals.     

The turbulent life of Sirevirus retrotransposons and the evolution of the maize genome: more than ten thousand elements tell the story

Sireviruses are one of the three genera of Copia long terminal repeat (LTR) retrotransposons, exclusive to and highly abundant in plants, and with a unique, among retrotransposons, genome structure. Yet, perhaps due to the few references to the Sirevirus origin of some families, compounded by the difficulty in correctly assigning retrotransposon families into genera, Sireviruses have hardly featured in recent research. As a result, analysis at this key level of classification and details of their colonization and impact on plant genomes are currently lacking. Recently, however, it became possible to accurately assign elements from diverse families to this genus in one step, based on highly conserved sequence motifs. Hence, Sirevirus dynamics in the relatively obese maize genome can now be comprehensively studied. Overall, we identified >10 600 intact and approximately 28 000 degenerate Sirevirus elements from a plethora of families, some brought into the genus for the first time. Sireviruses make up approximately 90% of the Copia population and it is the only genus that has successfully infiltrated the genome, possibly by experiencing intense amplification during the last 600 000 years, while being constantly recycled by host mechanisms. They accumulate in chromosome-distal gene-rich areas, where they insert in between gene islands, mainly in preferred zones within their own genomes. Sirevirus LTRs are heavily methylated, while there is evidence for a palindromic consensus target sequence. This work brings Sireviruses in the spotlight, elucidating their lifestyle and history, and suggesting their crucial role in the current genomic make-up of maize, and possibly other plant hosts.     

Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors

Glioblastoma, (grade IV astrocytoma), is characterized by rapid growth and resistance to treatment. Identification of markers of aggressiveness in this tumor could represent new therapeutic targets. Interleukins (IL)-6 and IL-10 may be considered as possible candidates, regulating cell growth, resistance to chemotherapy and angiogenesis. ELISPOT method provides a useful tool for the determination of the exact cell number of peripheral lymphocytes secreting a specific cytokine. IL-6 and IL-10 secretion levels were determined using ELISPOT methodology in peripheral blood mononuclear cells of 18 patients with astrocytic neoplasms (3 grade II and 15 grade IV), in parallel with 18 healthy controls. Additionally, immunohistochemical expression of these two cytokines was performed in paraffin-embedded neoplastic tissue in 12 of these patients. The secretion of IL-6 from peripheral monocytes was significantly higher in glioma patients compared to controls (P = 0.0003). In addition, IL-10 secretion from peripheral mononuclear and tumor cells of glioma patients was also higher as compared to healthy controls (P = 0.0002). Based on immunohistochemical staining, IL-6 expression was localized in tumor cells and macrophages as well as in areas of large ischemic necrosis, while the major source of IL-10 expression in glioblastomas was the microglia/macrophage cells. It is suggested that IL-10 contributes to the progression of astrocytomas by suppressing the patient’s immune response, whereas IL-6 provides an additional growth advantage. This study demonstrates for the first time the usefulness of ELISPOT in estimating the secretion of IL-6 and IL-10 from peripheral blood and the correlation of their expression in neoplastic cells. Christina Piperi and Penelope Korkolopoulou have equally contributed to this work.     

Towards a mechanistic understanding of reciprocal drug–microbiome interactions

Broad‐spectrum antibiotics target multiple gram‐positive and gram‐negative bacteria, and can collaterally damage the gut microbiota. Yet, our knowledge of the extent of damage, the antibiotic activity spectra, and the resistance mechanisms of gut microbes is sparse. This limits our ability to mitigate microbiome‐facilitated spread of antibiotic resistance. In addition to antibiotics, non‐antibiotic drugs affect the human microbiome, as shown by metagenomics as well as in vitro studies. Microbiome–drug interactions are bidirectional, as microbes can also modulate drugs. Chemical modifications of antibiotics mostly function as antimicrobial resistance mechanisms, while metabolism of non‐antibiotics can also change the drugs’ pharmacodynamic, pharmacokinetic, and toxic properties. Recent studies have started to unravel the extensive capacity of gut microbes to metabolize drugs, the mechanisms, and the relevance of such events for drug treatment. These findings raise the question whether and to which degree these reciprocal drug–microbiome interactions will differ across individuals, and how to take them into account in drug discovery and precision medicine. This review describes recent developments in the field and discusses future study areas that will benefit from systems biology approaches to better understand the mechanistic role of the human gut microbiota in drug actions.     

Biologic Treatment of Mild and Moderate Intervertebral Disc Degeneration

Disc degeneration is the most common cause of back pain in adults and has enormous socioeconomic implications. Conservative management is ineffective in most cases, and results of surgical treatment have not yet reached desirable standards. Biologic treatment options are an alternative to the above conventional management and have become very attractive in recent years. The present review highlights the currently available biologic treatment options in mild and moderate disc degeneration, where a potential for regeneration still exists. Biologic treatment options include protein-based and cell-based therapies. Protein-based therapies involve administration of biologic factors into the intervertebral disc to enhance matrix synthesis, delay degeneration or impede inflammation. These factors can be delivered by an intradiscal injection, alone or in combination with cells or tissue scaffolds and by gene therapy. Cell-based therapies comprise treatment strategies that aim to either replace necrotic or apoptotic cells, or minimize cell death. Cell-based therapies are more appropriate in moderate stages of degenerated disc disease, when cell population is diminished; therefore, the effect of administration of growth factors would be insufficient. Although clinical application of biologic treatments is far from being an everyday practice, the existing studies demonstrate promising results that will allow the future design of more sophisticated methods of biologic intervention to treat intervertebral disc degeneration.