Morphological,biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of β-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host–bacteria relationship.     

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

Biodegradable polymers from renewable sources: rheological characterization of hemicellulose-based hydrogels

Hemicellulose-based hydrogels were prepared by radical polymerization of 2-hydroxyethyl methacrylate or poly(ethylene glycol) dimethacrylate with oligomeric hydrosoluble hemicellulose modified with well-defined amounts of methacrylic functions. The polymerization reaction was carried out in water at 40 degrees C using a redox initiator system. The hydrogels were in general elastic, soft, and easily swellable in water. Their viscoelastic properties were determined by oscillatory shear measurements on 2 mm thick hydrogels under a slight compression to avoid slip, over the frequency range 10(-1) to 10(2). The rheological characterization indicated that the elastic response of the hydrogels was stronger than the viscous response, leading to the conclusion that the hydrogel systems displayed a predominantly solid-like behavior. The curves showed an increase in shear storage modulus with increasing cross-linking density. The nature of the synthetic comonomer in the hemicellulose-based hydrogels also influenced the shear storage modulus. Comparison of hemicellulose-based hydrogels with pure poly(2-hydroxyethyl methacrylate) hydrogels showed that their behaviors were rather similar, demonstrating that the synthetic procedure made it possible to prepare hemicellulose-based hydrogels with properties similar to those of pure poly(2-hydroxyethyl methacrylate) hydrogels.     

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 ± 20.8 and 192 ± 17.6 mm Hg, respectively, versus 213 ± 18 mm Hg in hypertensive controls; both p < 0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p > 0.05), prevented the vascular remodeling found in 2K-1C rats (all p < 0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p < 0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p < 0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.     

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.     

PULSES OF PHOSPHATE PROMOTE DOMINANCE OF THE TOXIC CYANOPHYTE CYLINDROSPERMOPSIS RACIBORSKII IN A SUBTROPICAL WATER RESERVOIR1

The role of dissolved inorganic phosphorus (DIP) in promoting dominance of the toxic nitrogen (N)‐fixing cyanobacterium Cylindrospermopsis raciborskii (Wo?osz.) Seenayya et Subba Raju was examined in a subtropical water reservoir, Lake Samsonvale (=North Pine reservoir). A novel in situ bioassay approach, using dialysis tubing rather than bottles or bags, was used to determine the change in C. raciborskii dominance with daily additions of DIP. A statistically significant increase in dominance of C. raciborskii was observed when DIP was added at two concentrations (0.32 μM and 16 μM) in a daily pulse over a 4 d period in three separate experiments in the summer of 2006/2007. There was an increase in both C. raciborskii cell concentrations and biovolume in two DIP treatments, but not in the ammoniacal N + DIP treatment. In addition, overall phytoplankton cell concentrations increased with DIP addition, indicating that Lake Samsonvale was DIP limited at the time of experiments. Given the bioassay response, it is likely that dominance of C. raciborskii could increase in Lake Samsonvale with periodic injections of DIP such as inflow events.     

Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.     

Synthesis and structure-activity relationship of 7-azaindole piperidine derivatives as CCR2 antagonists

The synthesis and structure-activity relationship of a series of 7-azaindole piperidine derivatives are described. SAR studies led to the discovery of the potent CCR2 antagonists displaying IC(50) values in the nanomolar range. The representative compound 15 showed reasonable P450 and pharmacokinetics profile.