Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Human prolidase and prolidase deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations

Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the prolidase deficiency (PD). Prolidase deficiency is a rare recessive disorder caused by mutations in the prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.     

Folding propensity and biological activity of peptides: the effect of a single stereochemical isomerization on the conformational properties of bombinins in aqueous solution

Folding propensities of bombinins H2 and H4, two members of amphibian bombinins H, a family of 17-20 residue alpha-helical peptides, have been investigated by means of circular dichroism (CD) measurements and molecular dynamics (MD) simulations. The two peptides, with primary structure IIGPVLGLVGSALGGLLKKI-NH2 and differing only for the configuration of the second aminoacid (an L-isoleucine in H2 and a D-alloisoleucine in H4) behave rather differently in solution. In particular both CD measurements and MD simulations indicate that bombinin H2 shows a markedly higher tendency to fold. From a careful inspection of MD trajectories it emerges that the stereochemical isomerization mutation of residue 2 to D-alloisoleucine in H4 peptide, drastically decreases its ability to form intrapeptide contacts. MD simulations also indicate that the conformational sampling in both systems derives from a subtle combination of energetic and entropic effects both involving the peptide itself and the solvent. The present results have been finally paralleled with preliminary information on bombinins H2 and H4 biological activity, i.e. interaction with membrane, supporting the hypothesis of an "already folded" conformation in water rather than interfacial folding tenet.     

Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice

The purpose of this study was to determine the effect of exercise on wound healing and inflammation in young (3 mo) and old (18 mo) female BALB/cByJ mice. Mice were assigned to either exercise or sedentary control (control) groups. The exercise group mice were run on a motorized treadmill at a moderate intensity 30 min/day for 8 days. All mice were given four full-thickness dermal wounds, and the rate of wound closure was assessed daily for 10 days. Four months later, the aged mice were rerandomized to treatment, wounded again in different locations, and wounds were harvested at 1, 3, or 5 days postwounding. Wound tissue was analyzed for IL-1beta, IL-6, keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), and TNF-alpha protein. Myeloperoxidase (MPO) activity and F4/80 mRNA were assessed as an indirect measure of neutrophil and macrophage content, respectively. There was a trend (P = 0.10) for exercise to reduce wound size in young mice, and exercise significantly (P < 0.05) decreased wound size in old mice. TNF-alpha, KC, and MCP-1 were significantly (P < 0.05) lower in wounds from exercised old mice compared with control. No group differences were found for wound IL-1beta or IL-6, MPO activity, or F4/80 mRNA. Our data suggest that exercise accelerates the wound healing process in old mice. This improved healing response in the old mice may be the result of an exercise-induced anti-inflammatory response in the wound.     

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.     

Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.     

Consequences of refuge for the functional response of Dermestes ater (Coleoptera: Dermestidae) to Musca domestica (Diptera: Muscidae)

It is well known that a predator has the potential to regulate a prey population only if the predator responds to increases in prey density and inflicts greater mortality rates. Predators may cause such density-dependent mortality depending on the nature of the functional and numerical responses. Yet, few studies have examined the relationship between the addition of refuges and the characteristic of functional response fits. We investigated whether addition of a refuge changed the type of functional response exhibited by Dermestes ater on Musca domestica, comparing the inherent ability of D. ater to kill houseflies in the absence and in the presence of refuge. An additional laboratory experiment was also carried out to assess handling and searching times exhibited by D. ater. Logistic regression analyses revealed a type III functional response for predator–prey interaction without refuge, and results were described by the random predator equation. The mean number of prey killed did not differ between experimental habitats, indicating that the addition of refuge did not inhibit predation. However, predators that interacted with prey without refuge spent less time searching for prey at higher densities, increasing predatory interaction. We concluded that this interaction may be weak, because data from experiments with refuge fitted poorly to models. However, the high variability and the nonsignificance of the data from the experiment with refuge show the importance of refuge for promoting spatial heterogeneity, which may prevent prey extinction.     

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ß-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.