Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.     

Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.     

Ecosystem changes associated with grazing in subhumid South American grasslands

Question: What are the changes in vegetation structure, soil attributes and mesofauna associated with grazing in mesic grasslands? Location: Southern Campos of the Río de la Plata grasslands, in south‐central Uruguay. Methods: We surveyed seven continuously grazed and ungrazed paired plots. Plant and litter cover were recorded on three 5‐m interception lines placed parallel to the fence in each plot. We extracted soil fauna from a 10 cm deep composite sample and analysed the oribatids. Soil attributes included bulk density, water content, organic carbon (in particulate and mineral associated organic matter) and nitrogen content and root biomass at different depths. Changes in floristic, Plant Functional Types and mesofauna composition were analysed by Non‐metric Multidimensional Scaling. Results: Species number was lower in ungrazed than in grazed plots. Of 105 species in grazed plots only three were exotics. Shrub and litter cover were significantly higher inside the exclosures, while the cover of Cyperaceae‐Juncaceae was lower. Grazing treatments differed significantly in plant and oribatid species composition. Grazing exclusion significantly reduced soil bulk density and increased soil water content. Carbon content in particulate organic matter was lower in the upper soil of ungrazed sites, but deeper in the profile, grazing exclosures had 8% more carbon in the mineral associated organic matter. Conclusions Our results generally agree with previous studies but deviate from the results of previous analyses in (1) the increase of shrub cover in ungrazed sites; (2) the redistribution of the soil organic carbon in the profile and (3) the low invasibility of the prairies regardless of grazing regime.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Stimulation effect of lithium on the metabolic activity of liver tissue mitochondria measured by microcalorimetry

The effect of Li(I) on the metabolism of mitochondria isolated from Carassius auratus liver tissue was investigated by microcalorimetric method to provide evidence for mitochondria hypothesis of biporlar disorder (BPD) and to explore therapeutic mechanism of drug for treatment of BPD. Obvious stimulation induced by Li(I) on mitochondria metabolism was reflected by power-time (P-t) curves. The power-time curves of hepatic mitochondria metabolism without Li(I) could be divided into four parts: lag phase, active recovery phase, stationary phase, and decline phase. When Li(I) was added, the second heat peak occurred in a concentration-dependent sequence. Considering the first heat peak on the p-t curves, Li(I) in the range of therapeutic and lower concentration induced slight alterations in comparison with the characteristic heat peak observed in the control. However, Li(I) above the therapeutic concentration resulted in significant changes. Heat output increased with the concentration of Li(I), but the rate constant (k ₂) and the maximum heat power (P _max2) for the second heat peak reached maximum value in the range of therapeutic concentration. Mechanism of activation of mitoKatp was suggested and discussed.     

Seven day oral supplementation with Cardax TM (disodium disuccinate astaxanthin) provides significant cardioprotection and reduces oxidative stress in rats

In the current study, the improved oral bioavailability of a synthetic astaxanthin derivative (Cardax^TM; disodium disuccinate astaxanthin) was utilized to evaluate its potential effects as a cardioprotective agent after 7-day subchronic oral administration as a feed supplement to Sprague-Dawley rats. Animals received one of two concentrations of Cardax^TM in feed (0.1 and 0.4%; ∼125 and 500 mg/kg/day, respectively) or control feed without drug for 7 days prior to the infarct study carried out on day 8. Thirty minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 h of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percentage (%) of the area at risk (AAR; IS/AAR,%) of 61 ± 1.8%. The AAR was quantified by Patent blue dye injection, and IS was determined by triphenyltetrazolium chloride (TTC) staining. Cardax^TM at 0.1 and 0.4% in feed for 7 days resulted in a significant mean reduction in IS/AAR,% to 45 ± 2.0% (26% salvage) and 39 ± 1.5% (36% salvage), respectively. Myocardial levels of free astaxanthin achieved after 7-day supplementation at each of the two concentrations (400 ± 65 nM and 1634 ± 90 nM, respectively) demonstrated excellent solid-tissue target organ loading after oral supplementation. Parallel trends in reduction of plasma levels of multiple lipid peroxidation products with disodium disuccinate astaxanthin supplementation were observed, consistent with the documented in vitro antioxidant mechanism of action. These results extend the potential utility of this compound for cardioprotection to the elective human cardiovascular patient population, for which 7-day oral pre-treatment (as with statins) provides significant reductions in induced periprocedural infarct size.     

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of alpha-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-alpha, IL-1beta, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.