The Effect of Matrix Metalloproteinase Inhibition on Intracellular Signaling in Adipogenic Differentiation of 3T3-L1 Cells

The growing obesity epidemic has led to an increased interest in the study of adipogenesis, which can be modeled in mouse 3T3-L1 fibroblast cells. Extracellular matrix remodeling is a critical process during adipogenesis that is facilitated by matrix metalloproteinases (MMPs). To better understand the function of MMPs, the MMP inhibitor Ilomostat was used on 3T3-L1 preadipocytes along with rosiglitazone, a drug that upregulates the transcription factor peroxisome-proliferator-activated receptor γ (PPARγ). We predicted that if MMP inhibition prevents activation of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ), the effect of Ilomostat should overcome the upregulation of downstream PPARγ by rosiglitazone. Results suggest that MMP inhibition prevents adipogenic conversion despite the addition of the PPARγ agonist, and that PPARγ expression was slightly decreased in cells treated with both rosiglitazone and Ilomostat. These results will help clarify the downstream effects of MMPs during adipogenesis and provide more insight into specific targets for obesity treatment

Neonicotinoid trapping by the FA1 site of human serum albumin

AbstractNeonicotinoids are a widely used class of insecticides that target the acetylcholine recognition site of the nicotinic acetylcholine receptors in the central nervous system of insects. Although neonicotinoids display a high specificity for insects, their use has been recently debated since several studies led to the hypothesis that they may have adverse ecological effects and potential risks to mammals and even humans. Due to their hydrophobic nature, neonicotinoids need specific carriers to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key carrier of endogenous and exogenous compounds. The in silico docking and ligand binding properties of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam to HSA are here reported. Neonicotinoids bind to multiple fatty acid (FA) binding sites, preferentially to the FA1 pocket, with high affinity. Values of the dissociation equilibrium constant for neonicotinoid binding FA1 of HSA (i.e., calcKn) derived from in silico docking simulations (ranging between 3.9 × 10−5 and 6.3 × 10−4 M) agree with those determined experimentally from competitive inhibition of heme‐Fe(III) binding (i.e., expKn; ranging between 2.1 × 10−5 and 6.9 × 10−5 M). Accounting for the HSA concentration in vivo (~7.5 10−4 M), values of Kn here determined suggest that the formation of the HSA:neonicotinoid complexes may occur in vivo. Therefore, HSA appears to be an important determinant for neonicotinoid transport and distribution to tissues and organs, particularly to the liver where they are metabolized

Hypoalbuminemia as a predictor of acute kidney injury during colistin treatment

This study aimed to assess the predictors of acute kidney injury (AKI) during colistin therapy in a cohort of patients with bloodstream infections (BSI) due to colistin-susceptible Gram-negative bacteria, focusing on the role of serum albumin levels. The study consisted of two parts: (1) a multicentre retrospective clinical study to assess the predictors of AKI during colistin therapy, defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria; and (2) bioinformatic and biochemical characterization of the possible interaction between human serum albumin and colistin. Among the 170 patients included in the study, 71 (42%), 35 (21%), and 11 (6%) developed KDIGO stage 1 (K1-AKI), KDIGO stage 2 (K2-AKI), and KDIGO stage 3 (K3-AKI), respectively. In multivariable analyses, serum albumin <2.5 g/dL was independently associated with K1-AKI (subdistribution hazard ratio [sHR] 1.85, 95% confidence interval [CI] 1.17\u20132.93, p = 0.009) and K2-AKI (sHR 2.37, 95% CI 1.15\u20134.87, p = 0.019). Bioinformatic and biochemical analyses provided additional information nurturing the discussion on how hypoalbuminemia favors development of AKI during colistin therapy. In conclusion, severe hypoalbuminemia independently predicted AKI during colistin therapy in a large cohort of patients with BSI due to colistin-susceptible Gram-negative bacteria. Further study is needed to clarify the underlying causal pathways

In situ enrichment of ocean crust microbes on igneous minerals and glasses using an osmotic flow-through device

Author Posting. © American Geophysical Union, 2011. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geochemistry Geophysics Geosystems 12 (2011): Q06007, doi:10.1029/2010GC003424.The Integrated Ocean Drilling Program (IODP) Hole 1301A on the eastern flank of Juan de Fuca Ridge was used in the first long-term deployment of microbial enrichment flow cells using osmotically driven pumps in a subseafloor borehole. Three novel osmotically driven colonization systems with unidirectional flow were deployed in the borehole and incubated for 4 years to determine the microbial colonization preferences for 12 minerals and glasses present in igneous rocks. Following recovery of the colonization systems, we measured cell density on the minerals and glasses by fluorescent staining and direct counting and found some significant differences between mineral samples. We also determined the abundance of mesophilic and thermophilic culturable organotrophs grown on marine R2A medium and identified isolates by partial 16S or 18S rDNA sequencing. We found that nine distinct phylotypes of culturable mesophilic oligotrophs were present on the minerals and glasses and that eight of the nine can reduce nitrate and oxidize iron. Fe(II)-rich olivine minerals had the highest density of total countable cells and culturable organotrophic mesophiles, as well as the only culturable organotrophic thermophiles. These results suggest that olivine (a common igneous mineral) in seawater-recharged ocean crust is capable of supporting microbial communities, that iron oxidation and nitrate reduction may be important physiological characteristics of ocean crust microbes, and that heterogeneously distributed minerals in marine igneous rocks likely influence the distribution of microbial communities in the ocean crust.The subseafloor flow cell enrichment chambers were funded by a small grant from the Ocean Drilling Program. This work was also funded by NASA grant NNX08AO22G, NSF OCE 0727119 to C.G.W., NSF OCE 0452333 to S.M.S., and OCE‐0550713 and OCE‐0727952 to A.T.F., PSU, and OSU

Design and analysis of algorithms for dynamic allocation of different permutation zones in WiMAX networks

WiMAX (Worldwide Interoperability for Microwave Access) , based on the IEEE 802.16 standard, defines wireless networks combining key characteristics of wide-area cellular networks as well as short-range networks, namely mobility and high data throughput. The current Mobile WiMAX technology is mainly based on the IEEE 802.16e amendment, which specifies the Orthogonal Frequency Division Multiple Access (OFDMA) air interface and provides support for mobility. IEEE 802.16e OFDMA standard provides for subchannelization techniques as a means to better manage network performance to address specific coverage and capacity requirements. Subchannels may be constituted using different permutation schemes to exploit frequency diversity or frequency selectivity. A number of contiguous OFDMA symbols, in the downlink frame or the uplink frame, that use the same permutation scheme constitute a permutation zone. The Down Link (DL) subframe or the Up Link (UL) subframe may contain more than one permutation zone. The first section of this work analyses the main issues connected with the presence and the management of multiple permutation zones within the DL WiMAX subframe. In the second section some possible multi-zone scheduling algorithms are proposed and implemented. The final section analyses and evaluates the performance of various schedulers. The appendix reports the work done for implementing support to SUB-DL-UL-MAP MAC messages within the ns-2 WiMAX simulator, and analyses the improvements deriving from their use

A possible molecular mechanism for parasitic inhibition by lactoferrin

Transferrins (Tfs) belong to a family of iron-binding glycoproteins possessing similar aminoacid sequence though they have different biological functions and locations. Lactoferrin (Lf) is expressed and secreted from glandular epithelial cells and from mature neutrophiles of mammalian and it is an important component of the aspecific host defence or natural immunity, including resistance to parasitic infections. Serum transferrin (sTf) is synthesized by the liver of mammals and secreted into the blood stream; its primary function is iron transport. Ovotransferrin (Otrf), synthesized by avians, displays both iron transport and protective functions. Parasites synthesize papain-like cysteine proteases that are relevant for the virulence and pathogenicity of parasites, being involved in several aspects of the parasite life cycle, it is therefore possible that the antiparasitic activity of Lf could be due to the inhibition of parasitic papain-like cysteine protease that we have recently observed. In this study we have investigated the thermodynamic parameters of hLf, bLf and Otrf inhibition of the parasitic papain-like type I cysteine proteases from Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei. bLf, hLf and Otrf, both in the apo- and olo-forms, showed time- and concentration-dependent inhibition of the catalytic activity of papain and of type I proteases from L.infantum, T. cruzi and T. brucei. The KI values observed for bLf and hLf inhibition of L. infantum, T. cruzi and T. brucei proteases were in the nanomolar range (KI = 3.1 nM), lower than KI values observed for papain inhibition (KI = 24 nM). Otrf showed lower inhibition of cysteine proteases (KI = 0.6 µM). On the contrary, sTf did not display any inhibition towards parasitic proteases, according to its different role in mammals. The inhibition of parasitic cysteine proteases by hLf, bLf and Otrf appeared to conform to a competitive mechanism. The observed pH optimum for bLf inhibition of parasitic proteases was around neutrality, while it was acidic for hLf and alkaline for Otrf. The further quantitative analysis of pH dependence of the intrinsic ligand-independent inhibition constant KI allowed the evaluation of pKa values that define the acid-base equilibrium of amino acidic residue(s) modulating the enzyme(s)-inhibitor recognition events. SDS-PAGE showed that hLf, bLf and Otrf were easily degraded by either papain or parasitic type I protease during the assay incubation time (few minutes) and it is likely that one or more protease inhibitory peptides were generated from protein hydrolysis. As a matter of fact, a sequence present near the C-terminus of human (hLf) and bovine (bLf) lactoferrin shows homology with the sequence of the active site of cystatins, which are competitive inhibitors of papain-like cysteine proteases. The same sequence is present, though with lower homology, in Otrf and, with even lower homology, in sTf. Therefore, we have charactherized by MALDI-TOF the profile of Lf cleavage by papain and preliminary data suggest the presence of a cystatin-like peptide in two proteolytic fragments of hLf and in one proteolytic fragment of bLf.Le transferrine (Tfs) sono una famiglia di glicoproteine in grado di legare reversibilmente il ferro, e presentano un’elevata omologia di sequenza tra tutti i membri di questa famiglia. In particolare la lattoferrina (Lf) è prodotta e secreta nelle cellule ghiandolari epiteliali ed è presente nei granuli dei granulociti neutrofili dei mammiferi. Essa rappresenta un’importante componente della difesa immunitaria aspecifica dell’ospite. La siero transferrina (sTf) invece è sintetizzata nel fegato, ed è coinvolta unicamente nel trasporto del ferro. L’ovotransferrina (Otrf), l’omologa aviaria della Lf , svolge sia una funzione di difesa che di trasporto del ferro. Le proteasi a cisteina papaina-simili da protozoi e metazoi parassiti, secretorie e di membrana, partecipano ai processi di morfogenesi degli organismi patogeni, sono implicate nell’invasione delle cellule e dei tessuti da parte dei parassiti, riducono la risposta immunitaria dell’ospite e costituiscono uno dei fattori più rilevanti nelle patologie associate alle infestazioni da parassiti. Pertanto, a fronte della capacità della Lf di svolgere un’azione antiparassitaria, noi abbiamo ipotizzato che la Lf possa svolgere tale azione grazie all’inattivazione delle suddette proteasi a cisteina. Nel presente lavoro abbiamo studiato le proprietà termodinamiche dell’inibizione delle proteasi a cisteina di alcuni parassiti, quali Leishmania infantum, Tripanosoma cruzi e Trypanosoma brucei. Tale studio è stato esteso anche verso la papaina, capostipite di questa famiglia. Innanzitutto è emerso una maggiore affinità delle transferrine studiate (bLf, hLf e Otrf, in forma apo e olo) verso le proteasi parassitarie (KI = 3.1 nM) rispetto a quella verso la papaina (KI = 24 nM). Otrf risulta essere l’inibitore con minore affinità (KI = 0.6 µM). Inoltre è emerso che la sTf non è in grado di svolgere un’azione inibitoria, coerentemente con la sua funzione nei mammiferi. L’inibizione di tutte le proteasi risulta essere conforme ad un meccanismo di inibizione competitivo. Da un’analisi della dipendenza del pH dei suddetti fenomeni di inibizione si è osservato una maggiore attività della bLf a pH fisiologico mentre la hLf inibisce più efficacemente a valori di pH acidi (pH = 5.0) e la Otrf a valori di pH alcalini (pH = 9.0). La dipendenza dal pH del parametro KI relativo all’interazione degli inibitori studiati con gli enzimi, ha permesso di evidenziare la variazione di diversi valori di pKa, ascrivibili ad eventi di protonazione e deprotonazione di differenti gruppi ionizzabili. Inoltre si è osservato, a seguito di SDS-page, una parziale idrolisi della Lf e della Otrf durante diverse incubazioni con la papaina e con le proteasi parassitarie, e l’insieme di tali dati suggerisce la liberazione di un peptide, che può quindi inibire competitivamente le suddette proteasi a cisteina. A supporto di tale ipotesi, si è osservato in presenza della porzione C-terminale della Lf, una sequenza aminoacidica che presenta un’elevata omologia con il sito attivo delle cistatine, una (super)famiglia di proteina, riconosciute essere gli inibitori reversibili per eccellenza delle proteasi a cisteina papaina-simili. Abbiamo quindi caratterizzato, mediante MALDI TOF, il profilo di idrolisi della Lf ad opera della papaina, ed è stata riscontrata la presenza di frammenti contenenti tale sequenza cistatinosimile, sia nella bLf che nell’hLf

Reactivity of the human hemoglobin "dark side".

Ligand binding to the heme distal side is a paradigm of biochemistry. However, X-ray crystallographic studies highlighted the possibility that O2 and NO2- may bind to the proximal heme side of ferrous human hemoglobin (Hb) alpha-chains complexed with the alpha-hemoglobin stabilizing protein and to ferric human hemoglobin beta-chains, respectively. Strikingly, the role generally played by the proximal HisF8 residue is played by the distal HisE7 side chain forming the trans axial ligand of the hemeFe atom. This: i) brings to light that Hb may utilize both heme distal and proximal sides for ligand discrimination, ii) draws attention to the nonequivalence of alpha- and beta-chains, and iii) highlights the possibility that partially unfolded Hb derivatives may display transient ligand-binding properties different from those of the native globin