Potential immunoregulatory role of heme oxygenase-1 in human milk: a combined biochemical and molecular modeling approach

Human milk contains biological factors that are involved in a newborn's growth and immune system regulation. By integrating standard biochemical experimental protocols with computational methods, the present study investigates the presence of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in human milk at different levels of maturation and in milk formulae. Furthermore, we evaluated cytokine and glutathione S-transferase (GSH) levels. Samples were collected from colostrum (on Day 1 after birth), from transition milk (on Postdelivery Days 7 and 14) and from mature milk (on Day 30 after delivery) in 14 healthy women. HO-1 protein, GSH and cytokines levels were measured using enzyme-linked immunosorbent assay and flow cytometry. HO-1 protein levels were significantly higher in colostrum (1.33 ng/ml; 5th centile 0.92; 95th centile 2.38) and in transition milk at 14 days (0.97 ng/ml; 5th centile 0.87; 95th centile 1.45) than in mature milk (0.9 ng/ml; 5th centile 0.8; 95th centile 1.38). Levels of HO-1 in milk formulae were similar to those in colostrum. No significant differences in GSH content were observed in mature milk, transition milk and colostrum, whereas significantly higher GSH levels were observed in milk formulae. No significant levels of cytokines, with the exception of interleukin-8, were found. Computational studies on the possible interactions between HO-1 and CD91 were carried out by a battery of softwares, namely, GRAMM (version 1.03), DALI, CLUSTALW (version 2.0), PatchDock and FireDock, mutually counterchecking and validating each other. The computational results, the strong convergence (to the same “solution”) of which finally leads to an “experimental-like” character, showed that HO-1 may bind to CD91, thus suggesting its major role as a new chaperokine in immune response regulation. These findings, which connect and integrate biochemical data and computational data interpretation, represent a synergistic and powerful means of conducting biological research.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Hydraulic connections of leaves and fruit to the parent plant in Capsicum frutescens (hot pepper) during fruit ripening

Background and Aims

The hydraulic architecture and water relations of fruits and leaves of Capsicum frutescens were measured before and during the fruiting phase in order to estimate the eventual impact of xylem cavitation and embolism on the hydraulic isolation of fruits and leaves before maturation/abscission.

Methods

Measurements were performed at three different growth stages: (1) actively growing plants with some flowers before anthesis (GS1), (2) plants with about 50 % fully expanded leaves and immature fruits (GS2) and (3) plants with mature fruits and senescing basal leaves (GS3). Leaf conductance to water vapour as well as leaf and fruit water potential were measured. Hydraulic measurements were made using both the high-pressure flow meter (HPFM) and the vacuum chamber (VC) technique.

Key Results

The hydraulic architecture of hot pepper plants during the fruiting phase was clearly addressed to favour water supply to growing fruits. Hydraulic measurements revealed that leaves of GS1 plants as well as leaves and fruit peduncles of GS2 plants were free from significant xylem embolism. Substantial increases in leaf petiole and fruit peduncle resistivity were recorded in GS3 plants irrespective of the hydraulic technique used. The higher fraction of resistivity measured using the VC technique compared with the HPFM technique was apparently due to conduit embolism.

Conclusions

The present study is the first to look at the hydraulics of leaves and fruits during growth and maturation through direct, simultaneous measurements of water status and xylem efficiency of both plant regions at different hours of the day.     

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1

The mitochondrial intermediate-conductance Ca²⁺-activated K⁺ channel mtK_Ca3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtK_Ca3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtK_Ca3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type K_V1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861–14866). A specific K_Ca3.1 inhibitor however did not have any detectable effect on cell proliferation or death.     

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

Recent studies have demonstrated that tocotrienol (T3) is superior to tocopherol (Toc) for cancer chemoprevention. However, there is little information on whether Toc influences the anticancer property of T3. In this study, we investigated the influence of Toc on the cytotoxic effects of δ-T3 in DLD-1 human colorectal adenocarcinoma cells. Toc, especially α-Toc, attenuated δ-T3-induced cytotoxicity and apoptosis in DLD-1 cells, whereas Toc alone did not exhibit any cytotoxic effect. δ-T3-induced cell cycle arrest and proapoptotic gene/protein expression (e.g., p21, p27, and caspases) were abrogated by α-Toc. Furthermore, coadministration of α-Toc decreased δ-T3 uptake into DLD-1 cells in a dose-dependent manner. These results indicate that α-Toc is not only less cytotoxic to cancer cells, but it also reduces the cytotoxicity of δ-T3 by inhibiting its cellular uptake.     

Species’ response patterns to habitat fragmentation: do trees support the extinction threshold hypothesis?

In fragmented landscapes the relationship between the probability of occurrence of single species and the amount of suitable habitat is usually not proportional, with a threshold habitat level below which the population becomes extinct. Ecological theory predicts that, although the reduction in species’ occurrence probabilities (and eventually the extinction threshold) is a direct consequence of habitat loss, habitat fragmentation might reduce species’ occurrence probabilities and affect the location of this threshold by reducing its predicted occurrence to lower levels of habitat amount. However, little is known about the validity of this extinction threshold hypothesis. Here, we performed analyses on the relationships between the probability of occurrence of eight tree species and the availability of forest habitat for two different empirical scenarios of low and moderate to high fragmentation. We partitioned the effects of habitat amount versus fragmentation by using two metrics: (1) the percentage of forest cover, and (2) the proportion of this percentage occurring in the largest forest patch. We find that, although decreasing forest cover had negative effects on the occurrence of tree species irrespective of fragmentation levels, forest fragmentation significantly modified the response pattern in six tree species, although only one species confirmed the extinction threshold hypothesis, which we interpret as a consequence of high degree of forest specialism and low dispersal ability. For most species, fragmentation either had positive effects or did not affect significantly the species’ probability of occurrence. This indicates that the effects of habitat fragmentation on tree species are weak relative to the effects of habitat amount, which is the main determinant of the reduction in species’ occurrence probabilities, and eventually species extinction, in fragmented landscapes.     

Novel imidazobenzazepine derivatives as dual H1/5-HT2A antagonists for the treatment of sleep disorders

A novel imidazobenzazepine template (5a) with potent dual H₁/5-HT_2A antagonist activity was identified. Application of a zwitterionic approach to this poorly selective and poorly developable starting point successfully delivered a class of high quality leads, 3-[4-(3-R¹-2-R-5H-imidazo[1,2-b][2]benzazepin-11-yl)-1-piperazinyl]-2,2-dimethylpropanoic acids (e.g., 9, 19, 20, and 21), characterized by potent and balanced H₁/5-HT_2A receptor antagonist activities and good developability profiles.     

N-Substituted acetamidines and 2-methylimidazole derivatives as selective inhibitors of neuronal nitric oxide synthase

A series of N-substituted acetamidines and 2-methylimidazole derivatives structurally related to W1400 were synthesized and evaluated as Nitric Oxide Synthase (NOS) inhibitors. Analogs with sterically hindering isopropyl and phenyl substituents on the benzylic carbon connecting the aromatic core of W1400 to the acetamidine nitrogen, showed good inhibitory potency for nNOS (IC(50)=0.2 and 0.3 μM) and selectivity over eNOS (500 and 1166) and to a lesser extent over iNOS (50 and 100). A molecular modeling study allowed to shed light on the effects of the structural modifications on the selectivity of the designed inhibitors toward the different NOS isoforms.     

Discovery and structure–activity relationship of a novel spirocarbamate series of NPY Y5 antagonists

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3 mg/kg po.