Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Stochastic determinants of assemblage patterns in coral reef fishes: a quantification by means of two models

Synopsis One perspective emphasizing the importance of stochastic processes in determining coral reef fish assemblages implies that there is little organization in species richness, abundance structure, and spatial distribution. We examine the degree to which this perspective is correct by analyzing distribution of fishes on a collection of patch reefs (Discovery Bay, Jamaica). We ask the question whether these patches accumulate species and individuals in a manner consistent with stochastic expectations. To address this question we use two conceptual models, each permitting a different insight. One assumes that fish are distributed stochastically on patches while the other assumes presence of restrictions on fish distribution due to habitat structure. For each conceptual model we use two types of benchmark: we compare observed patterns to those predicted by theoretical models, and we also compare observed patterns to those obtained from a random reallocation of fish individuals to patches. We found that the conceptual model assuming stochastic processes appeared to provide weaker explanation of patterns than the conceptual model that includes restrictions due to habitat structure. Further, and more importantly, we found that (i) the community is shaped by a mixture of stochastic and non-stochastic mechanisms, and (ii) the stochastic assembly processes decrease in importance for species restricted to fewer microhabitat types and sites. Our study therefore indicates that patches do accumulate individuals and species in a manner consistent with stochastic expectations, however, this applies primarily to the habitat generalists (unrestricted species). By the same token, increased habitat specialization by some species imposes constraints on the stochastic model such that it eventually fails.     

Predicting the occurrence of persistent hotspots in ecosystem variables

Ecological resources and services (e.g. organisms, nutrient cycling) are distributed heterogeneously across landscapes. While spatial variation has been studied extensively, the pattern of hotspots and coolspots persisting over time – called persistent spatial variation (PSV) – has not. Yet this pattern imparts key information to managers about whether resources will be found consistently in certain locations or vary unpredictably. Anticipating whether an ecosystem variable will display PSV is thus a valuable prospect. We tested the ability of attributes of variables (e.g. niche breadth, abundance, temporal scale) to predict the occurrence of PSV. Using a new measure of PSV based on the F‐value of analysis of variance, we were able to 1) decompose the pattern of persistent hotspots into spatial and temporal components – ‘spatial variation’ of site mean values and ‘stability’ of time series at each site – and 2) identify predictors of these patterns in temperate lakes and tropical coastal rock pools. We found PSV to be highly predictable (R2 = up to 0.80) from an estimate of stability taken at a single site, as well as from other factors related to stability. These factors included whether the variable was environmental (stable, slow) or was an aggregate of other variables (stabilized by statistical averaging). Species properties like niche position and abundance were modest predictors because they correlated with PSV components of site occupancy, spatial variation and stability. We conclude that PSV and the distribution of resources in space and time might be predicted from simple temporal indicators (e.g. stability at a single location) when data are scarce.     

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π₀ = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E′, to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E′= 715 mN/m).     

Anchorage-dependent chick embryo fibroblasts synthesise DNA and proliferate when cultured in multilayered sheets suspended among glass fibres.

Chick embryo fibroblasts (CEFs) spontaneously form multicellular and multilayered sheets suspended on the network of glass fibres which are stabilized by fibronectin containing protein deposits located at cell-to-cell contacts. The cells situated within the sheets are surrounded by the neighbouring cells and their mechanical equilibrium is stabilised by intercellular "parabaric" effects. It was found that CEFs in the sheets retain relatively high mitotic activity corresponding to that observed in sparse monolayer cultures. These cells grew up to much higher local density than in confluent and contact-inhibited monolayer cultures and developed an abundance of microfilament bundles that terminated at vinculin-containing protein complexes. The results presented demonstrate that direct contact with solid substratum, cell-to-cell contacts, local cell density, and intercellular exchange of humoral factors are not directly involved in the density-dependent inhibition of growth observed in monolayer cultures. They also support the concepts concerning the role of mechanical equilibrium of cell membrane and sub-membranous cytoskeleton in the regulation of proliferation of non-transformed cells.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.     

A Structured Interface to the Object-Oriented Genomics Unified Schema for XML-Formatted Data

Data management systems are fast becoming required components in many biology laboratories as the role of computer-based information grows. Although the need for data management systems is on the rise, their inherent complexities can deter the full and routine use of their computational capabilities. The significant undertaking to implement a capable production system can be reduced in part by adapting an established data management system. In such a way, we are leveraging the Genomics Unified Schema (GUS) developed at the Computational Biology and Informatics Laboratory at the University of Pennsylvania as a foundation for managing and analysing DNA sequence data in centromere research projects around Arabidopsis thaliana and related species. Because GUS provides a core schema that includes support for genome sequences, mRNA and its expression, and annotated chromosomes, it is ideal for synthesising a variety of parameters to analyse these repetitive and highly dynamic portions of the genome. Despite this, production-strength data management frameworks are complex, requiring dedicated efforts to adapt and maintain. The work reported in this article addresses one component of such an effort, namely the pivotal task of marshalling data from various sources into GUS. In order to harness GUS for our project, and motivated by efficiency needs, we developed a structured framework for transferring data into GUS from outside sources. This technology is embodied in a GUS object-layer processor, XMLGUS. XMLGUS facilitates incorporating data into GUS by (i) formulating an XML interface that includes relational database key constraint definitions, (ii) regularising traversal through that XML, (iii) realising automatic processing of the XML with database key constraints and (iv) allowing for special processing of input data within the framework for automated processing. The application of XMLGUS to production pipeline processing for a sequencing project and inputting the Arabidopsis genome into GUS is discussed. XMLGUS is available from the Flora website (http://flora.ittc.ku.edu/).