Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs

Cytokinin oxidases (CKOs) play a major role in the regulation of hormone levels in plants by irreversibly degrading cytokinins. Two new cDNAs from maize (CKO2 and CKO3) were cloned and CKO activity of a recombinant CKO3 enzyme was demonstrated. CKO2 and CKO3 encode flavoproteins with 93% identity among each other compared with 45% identity with CKO1. The respective genes were mapped to BIN 3.05/06 and BIN 8.06 which belong to duplicated regions of the maize genome. For a better understanding of the role of CKO2 and CKO3 in maize development, their expression profiles were analysed in different organs and during kernel development via semi-quantitative RT-PCR. Different spatial and temporal expression patterns were observed for the two genes, as well as for CKO1 and two additional genes CKO4 and CKO5. CKO2 to CKO5 genes were mainly expressed in vegetative tissues, with unique expression patterns. CKO1 was most strongly expressed in the kernel. All five genes were expressed at early stages of kernel development, a period when a peak in cytokinin levels and a high cell division rate in the endosperm have been described. However, each gene had its own expression profile with a major difference concerning the onset of expression.     

Iron starvation leads to oxidative stress in Anabaena sp. strain PCC 7120

We establish here that iron deficiency causes oxidative stress in the cyanobacterium Anabaena sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.     

Anti-DNA antibodies: structure and pathogenic role

Anti DNA antibodies are generally classified into two major groups: Anti ds DNA (anti double .. stranded DNA) antibody and anti ss-DNA (anti single-stranded DNA) antibody. Anti-ds DNA antibodies are highly specific to systemic lupus erythematosus (SLE) and are probably involved in the pathogenesis of lupus nephritis. There are numerous serological tests for detecting anti-ds DNA. The detection of anti-ds DNA antibodies in the circulation of patients is one of the major criteria for the diagnosis of SLE; moreover, exacerbation of the disease are proceeded by increasing anti-DNA levels and the development of lupus nephritis, one of the most serious complications of the disease, strongly correlates with the presence of high avidity anti-DNA. It was reported that even normal individuals express anti-ds DNA. However, anti ds DNA found in healthy individuals is usually of the immunoglobulin M (IgM) isotype and shows a low affinity to ds-DNA. These natural antibodies are characterized by a wide cross-reactivity and are usually encoded by gene segments in the germ line configuration. In contrast, the ds-DNA antibodies involved influenced by various findings supporting the proposition that anti-ds DNA is involved in the pathogenesis of SLE, an enormous amount of scientific investigation has failed to reveal the exact mechanism through which this occurs.     

Oxidative stress disrupts nitric oxide synthase activation in liver endothelial cells

Oxidative stress may mediate vascular disruption associated with a loss of endothelial nitric oxide synthase (eNOS) activity and a hypersensitivity to the constrictor effects of endothelin-1 (ET-1). We hypothesize that this is due, in part, to uncoupling of ET(B) receptors from eNOS activation. Thus, we tested whether oxidative stress (OS) affects liver vascular relaxation by reducing basal and ET-1-induced NO production. Primary sinusoidal endothelial cell cultures were pretreated with H(2)O(2) (25 microM) for 1 or 6 h before a 10-min ET-1 stimulation. OS resulted in a significant basal and ET-1-induced decrease in NO production. Acute OS increased the monomeric form of the inhibitory protein caveolin-1 (1.2 +/- 0.05 vs 0.9 +/- 0.02, p < 0.01) and increased the eNOS-caveolin association as determined by coimmunoprecipitation (1.24 +/- 0.04 vs 0.97 +/- 0.04, p < 0.05). ET-1 stimulation further exacerbated these effects. Subacute OS inhibited ET-1-induced eNOS phosphorylation of serine 1177 (activation residue) (1 +/- 0.07 vs 1.6 +/- 0.04, p < 0.05) and dephosphorylation of the inhibitory residue threonine 495 (1.5 +/- 0.08 vs 0.7 +/- 0.02, p < 0.01). Additionally subacute OS resulted in dissociation of eNOS from ET(B) (0.8 +/- 0.09 vs 1.2 +/- 0.06, p < 0.05). Our findings indicate that acute and subacute oxidative stress result in the inhibition of induced nitric oxide synthase activity through distinct mechanisms dependent on caveolin-1 inhibition, ET(B) dissociation, and eNOS phosphorylation.     

Distinct Molecular Strategies for Hox-Mediated Limb Suppression in Drosophila: From Cooperativity to Dispensability/Antagonism in TALE Partnership

The emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways.     

Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases

Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 μg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis.     

Involvement of IL17A,IL17F and IL23R Polymorphisms in Colorectal Cancer Therapy

IL23/IL17 pathway plays an important role in the development of inflammatory bowel diseases (IBD). In general, the genes encoding the cytokines are genetically polymorphic and polymorphisms in genes IL23R and IL17 have been proved to be associated with its susceptibility to inflammatory diseases as well as cancer including colorectal cancer. Moreover, it has been shown that these interleukins are involved in anti-tumor or pro-tumor effects of various cancers. Previously, we showed that there is a significant association between IL17A, IL17F and IL23R polymorphisms as well as the occurrence of colorectal cancer and the clinical features of the disease. The purpose of the present work is to investigate an association between IL17A, IL17F and IL23R polymorphisms in 102 Tunisian patients with colorectal cancer treatment. The association was analyzed by statistical tools. We found that patients with mutated genotypes of IL17A G197A SNP could be a risk factor for the inefficiency of chemotherapy and radiotherapy. Unlike IL17F variant, patients with wild type genotypes require surgery and adjuvant chemotherapy. On the one hand, we found no evidence that supports a significant association between IL23R polymorphism and the combined genotypes of these three genes and the colorectal cancer treatment. On the other hand, we showed that there is an important interaction between IL17A/IL17F polymorphisms and the stage of the disease as well as its treatment. Finally, patients with IL17F wild type genotype highlighted that there is a valid longer OS without all treatments and with radiotherapy and a neoadjuvant chemotherapy. In contrast, we observed that there are no relationships between IL17A, IL23R and the survival of these patients neither with nor without the treatment. Our results suggest that polymorphisms in IL17A and IL17F genes may be a predictive source of colorectal cancer therapy type. Therefore, IL17F may serve as an independent prognostic factor for overall survival in patients with colorectal cancer.     

Multidrug-resistant phenotype and isolation of a Novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae

Background

ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species.

Results

E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla_SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla_SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein.

Conclusion

These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.     

Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.