Osteoporosis,inflammation and ageing

Osteoporosis is a condition characterized by low bone mass and increased bone fragility, putting patients at risk of fractures, which are major causes of morbidity substantially in older people. Osteoporosis is currently attributed to various endocrine, metabolic and mechanical factors. However, emerging clinical and molecular evidence suggests that inflammation also exerts significant influence on bone turnover, inducing osteoporosis. Numerous proinflammatory cytokines have been implicated in the regulation of osteoblasts and osteoclasts, and a shift towards an activated immune profile has been hypothesized as important risk factor. Chronic inflammation and the immune system remodelling characteristic of ageing, as well as of other pathological conditions commonly associated with osteoporosis, may be determinant pathogenetic factors. The present article will review the current perspectives on the interaction between bone and immune system in the elderly, providing an interpretation of osteoporosis in the light of inflamm-ageing.     

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+-dependent and tetrodotoxin-sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down-regulation of NOP receptors and activation of N/OFQ biosynthesis.     

Optimization of two-joint arm movements: a model technique or a result of natural selection?

The fossil record of early hominids suggests that their Arm length, and presumably stature and weight, had a tendency to increase. Using the minimum jerk principle and a related formulation of averaged specific power, ASP, with regard to selected two-joint Arm movements, the current paper explores relationships between ASP, hand trajectory length (or Arm length, or body mass) and mean movement speed, deriving relationships which indicate that ASP is proportional to cubic mean movement speed, but inversely proportional to hand trajectory length (or Arm length, or 1/3 power of body mass). Accordingly, an `ecological niche’ is modeled in a three-parameter space. Either ASP maximization for fixed movement time, or ASP minimization for fixed mean movement speed, taken as selective optimization criterion, allows the increasing of human Arm length during evolution, regardless of the arm-to-forearm length ratio.     

Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro

To determine whether IL-6 increases lipolysis and fat oxidation in patients with type 2 diabetes and/or whether it exerts this effect independently of changes to the hormonal milieu, patients with type 2 diabetes (D) and healthy control subjects (CON) underwent recombinant human (rh)IL-6 infusion for 3 h. Rates of appearance (Ra) and disappearance (Rd) of [U-(13C)]palmitate and [6,6-(2H2)]glucose were determined. rhIL-6 infusion increased (P < 0.05) palmitate Ra and Rd in a similar fashion in both groups. Neither plasma glucose concentration nor glucose Ra/Rd was affected by rhIL-6 infusion in either group, whereas rhIL-6 infusion resulted in a reduction (P < 0.05) in circulating insulin in D. Plasma growth hormone (GH) was increased (P < 0.05) by IL-6 in CON, and cortisol increased (P < 0.05) in response to IL-6 in both groups. To determine whether IL-6 was exerting its effect directly or through activation of these hormones, we performed cell culture experiments. Fully differentiated 3T3-L1 adipocytes were treated with PBS (control) IL-6, or IL-6 plus dexamethasone and GH. IL-6 treatment alone increased (P < 0.05) lipolysis, but this effect was reduced by the addition of dexamethasone and GH such that IL-6 plus dexamethasone and GH had blunted (P < 0.05) lipolysis compared with IL-6 alone. To assess whether IL-6 increases fat oxidation, L6 myotubes were treated with PBS (Control), IL-6, or AICAR, a compound known to increase lipid oxidation. Both IL-6 and AICAR markedly increased (P < 0.05) oxidation of [(14)C]palmitate compared with Control. Acute IL-6 treatment increased fatty acid turnover in D patients as well as healthy CON subjects. Moreover, IL-6 appears to be activating lipolysis independently of elevations in GH and/or cortisol and appears to be a potent catalyst for fat oxidation in muscle cells.     

Legionella waterline colonization: detection of Legionella species in domestic, hotel and hospital hot water systems

AIMS: An evaluation was made of the prevalence of Legionella species in hot water distribution systems in the city of Bologna (Italy) and their possible association with bacterial contamination (total counts and Pseudomonadaceae) and the chemical characteristics of the water (pH, Ca, Mg, Fe, Mn, Cu, Zn and Total Organic Carbon, TOC). METHODS AND RESULTS: A total of 137 hot water samples were analysed: 59 from the same number of private apartments, 46 from 11 hotels and 32 from five hospitals, all using the same water supply. Legionella species were detected in 40.0% of the distribution systems, L. pneumophila in 33.3%. The highest colonization was found in the hot water systems of hospitals (93.7% of samples positive for L. pneumophila, geometric mean: 2.4 x 10(3) CFU l(-1)), followed by the hotels (60.9%, geometric mean: 127.3 CFU l(-1)) and the apartments with centralized heating (41.9%, geometric mean: 30.5 CFU l(-1)). The apartments with independent heating systems showed a lower level of colonization (3.6% for Legionella species), with no evidence of L. pneumophila. Correlation analysis suggests that copper exerts an inhibiting action, while the TOC tends to favour the development of L. pneumophila. No statistically significant association was seen with Pseudomonadaceae, which were found at lower water temperatures than legionellae and in individual distribution points rather than in the whole network. CONCLUSIONS: The water recirculation system used by centralized boilers enhances the spreading of legionellae throughout the whole network, both in terms of the number of colonized sites and in terms of CFU count. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in Legionella colonization between types of buildings are not due to a variation in water supply but to other factors. Besides the importance of water recirculation, the study demonstrates the inhibiting action of copper and the favourable action of TOC on the development of L. pneumophila.     

Comparative structural analysis of low-molecular mass fragments of Rapana venosa hemocyanin obtained using two different procedures

Different fragments of the hemocyanin (Hc) isolated from the gastropod Rapana venosa containing a single functional unit (50 kDa), two functional units (100 kDa) and three functional units (150 kDa) were obtained in a dissociating buffer in the presence of Zn2+ and purified to homogeneity. Their conformations in solution were studied by means of small angle X-ray scattering (SAXS) and compared with those of the corresponding fragments previously obtained by limited proteolysis [Arch. Biochem. Biophys., 2000, 373, 154]. The overall shape of each fragment was determined using an ab initio approach. The crystal structures of the functional unit e from the same Hc and from another molluscan Hc (Octopus dofleini) were used to model 100 and 150 kDa fragments using rigid body movements to fit the corresponding SAXS patterns. Interesting differences were observed between the functional unit organization in the low-molecular mass fragments according to the two preparation methods, suggesting different localizations within the 11S functional subunit.     

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. I. Apo form

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contrast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C(1/2) 1.6-1.7 M. A similar two-state transition, with a slightly higher C(1/2) value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3-5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.     

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. II. Holo form

The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl<1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C(1/2)=1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C(1/2)=1.4 M). Cooperative transitions with C(1/2) values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C(1/2)=1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species.