Upregulation of MMPs in Metastatic Cascade of Breast Cancer To Brain

Background Brain metastasis is a lethal complication in triple negative breast cancer (TNBC) patients. Many factors including tumor cell molecular characteristics and biological environment are the main determinant in the brain metastasis process. Matrix metalloproteinases (MMPs) play a key role in extracellular matrix degradation, implicated in numerous aspects of metastasis processes of breast cancer. Methods After development of syngenic animal model of TNBC, primary breast cancer cells named 4T1T were isolated from tumor mass. Highly metastatic tumor cells named 4T1B were isolated and expanded from brain metastasis lesions of cancerous mice. Quantitative real-time polymerase chain reaction and gelatinase zymography were performed to analyze the expression of MMPs in transcriptomic and proteomic level in 4T1T and 4T1B. Results Our data revealed that, expression of MMPs was significantly upregulated in brain metastatic tumor cells. In transcriptomic level, MMP-2 and MMP-9 genes expression were up-regulated 4 and 3.4 folds in 4T1B, respectively. Zymographic analysis could be detect MMPs activity only in 4T1B. Conclusion These findings provided important insights regarding the gross alteration of MMPs expression in brain metastatic cascade of TNBC for the first time. Analysis of molecular properties of brain metastatic tumor cells can be used for understanding of molecular and genetic aspects of brain metastasis and also designing a targeted therapeutic strategies in combat with brain metastasis of TNBC.

Endogenous Enzymatic Activity in Dentin Treated with a Chitosan Primer

The aim of this study was to evaluate the effect of different concentrations of chitosan polymer on dentinal enzymatic activity by means of gelatin and in situ zymography. Human dentin was frozen and ground in a miller. Dentin powder aliquots were demineralized with phosphoric acid and treated with three different concentrations of lyophilized chitosan polymer (1, 0.5 and 0.1 wt%) dissolved in distilled water. Dentin proteins were extracted from each experimental group and electrophoresed under non-reducing conditions in 10% SDS-PAGE containing fluorescein-labeled gelatin. After 48 h in the incubation buffer at 37 °C, proteolytic activity was registered under long-wave UV light scanner and quantified by using Image J software. Furthermore, additional teeth (n = 4) were prepared for the in situ zymographic analysis in unrestored as well as restored dentin pretreated with the same chitosan primers. The registered enzymatic activity was directly proportional to the chitosan concentration and higher in the restored dentin groups (p < 0.05), except for the 0.1% chitosan primer. Chitosan 0.1% only showed faint expression of enzymatic activity compared to 1% and 0.5% concentrations. Chitosan 0.1% dissolved in water can produce significant reduction in MMPs activity and could possibly contribute to bond strength preservation over time.

Evaluation of tissue in repair with natural latex and / or hyaluronic acid in surgical bone defects

This study evaluated the bone repair in surgical defects of rats treated with hyaluronic acid (HA) associated or not with Hevea brasiliensis fraction protein (F-1). Bone defect were created in 15 albino Wistar rats divided into 3 groups (n=5): Control group (1) - blood clot; HA group (2) - 0.5% hyaluronic acid; HAF1 group (3) - 0.1% F-1 protein fraction dissolved in 0.5% hyaluronic acid. After 4 weeks, the animals were euthanized and the bone repair was evaluated through histomorphometric analysis, zymography and immunohistochemistry. The neoformed bone area did not show a significant difference (p = 0.757), but there was a tendency for bone trabeculation to increase in the groups HA and HAF1. For immunohistochemically analysis, there was a difference in vascular endothelial growth factor (VEGF) labeling (p = 0.023), being higher in the groups HA and HAF1 than the control group. No significant difference in bone sialoprotein (BSP) (p = 0.681), osteocalcin (p = 0.954), however, significant difference in platelet endothelial cell adhesion molecule-1 (CD-31) (p = 0.040), with HAF1 group being significantly lower than the control. For zymographic analysis, there was no significant difference for metalloproteinase-2 (MMP-2) (p = 0.068), but there was a tendency to increase MMP-2 in the HA group. Despite the influence on angiogenic factors and the apparent tendency for greater trabeculation in the HA and HAF1 groups, there was no significant difference in the area of newly formed bone tissue in the analyzed period.

Purification and Crystallographic Analysis of a Novel Cold-Active Esterase (HaEst1) from Halocynthiibacter arcticus

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 Å resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50–2.10 Å resolution. The unit cell parameters were a = 35.69 Å, b = 91.21 Å, c = 79.15 Å, and β = 96.9°.

Effect of Orally Administered Relaxin in Experimental Nash

BACKGROUND Relaxin (RLX), a hormone-like molecule with pleiotropic effects, has been found to reduce matrix deposition and mediate collagen degradation in animal models of chronic liver injuries and might be considered as an adjuvant therapeutic agent for progressive liver diseases. AIMS In the present study, we evaluated whether RLX affects the development of liver fibrosis in an experimental mouse model of NASH in C57BL6 male mice fed with a methionine-choline deficient diet (MCD). Methods Mice were treated per os as we intended to assess the enteric absorption and bioavailability of orally administered RLX (RLX purified from pig ovaries, IBSA SA, Lugano, Switzerland). Mice were fed the MCD diet for 6 weeks. After the initial 3 weeks, one group received drinking water supplemented with RLX (25 mcg/ml) whereas the other continued to receive regular water. A third group of mice fed a regular diet served as control. After 3 additional weeks mice were anesthetized and sacrificed. Results A significant, reduced expression of the pro-inflammatory cytokines TNF-&alpha; and of relevant markers of active fibrogenesis and extracellular matrix deposition, Col1A1 and &alpha;-SMA, was observed in mice treated with RLX vs controls. RLX also induced a significant decrease of TIMP1 and an increase of both MMP 2 and 9 when evaluated by zymographic analysis in liver extracts. Conclusions Although preliminary pharmacokinetics experiments showed barely detectable amounts of RLX in the blood of mice treated per os, these data support the absorption of orally administered RLX and confirm its potential therapeutic use in the management of liver fibrosis.

Identification and Characterization of a Cell Wall Hydrolase for Sporangiospore Maturation in Actinoplanes missouriensis

ABSTRACT The rare actinomycete Actinoplanes missouriensis grows as substrate mycelium and forms terminal sporangia containing a few hundred spores as dormant cells. Upon contact with water, the sporangia open up and release spores to external environments. Here, we report a cell wall hydrolase, GsmA, that is required for sporangiospore maturation in A. missouriensis. The gsmA gene is conserved among Actinoplanes species and several species of other rare actinomycetes. Transcription of gsmA is activated in the late stage of sporangium formation by the global transcriptional activator TcrA, which is involved in sporangium formation and dehiscence. GsmA is composed of an N-terminal signal peptide for the twin arginine translocation pathway, two tandem bacterial SH3-like domains, and a glucosaminidase domain. Zymographic analysis using a recombinant C-terminal glucosaminidase domain protein showed that GsmA is a hydrolase able to digest cell walls extracted from the vegetative mycelia of A. missouriensis and Streptomyces griseus. A gsmA deletion mutant (ΔgsmA) formed apparently normal sporangia, but they released chains of 2 to 20 spores under sporangium dehiscence-inducing conditions, indicating that spores did not completely mature in the mutant sporangia. From these results, we concluded that GsmA is a cell wall hydrolase for digesting peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in A. missouriensis. Unexpectedly, flagella were observed around the spore chains of the ΔgsmA mutant by transmission electron microscopy. The flagellar formation was strictly restricted to cell-cell interfaces, giving an important insight into the polarity of the flagellar biogenesis in a spherical spore. IMPORTANCE In streptomycetes, an aerial hypha is compartmentalized by multiple septations into prespores, which become spores through a series of maturation processes. However, little is known about these maturation processes. The rare actinomycete Actinoplanes missouriensis produces sporangiospores, which are assumed to be formed also from prespores generated by the compartmentalization of intrasporangium hyphae via septation. The identification of GsmA as a cell wall hydrolase for the separation of adjacent spores sheds light on the almost unknown processes of sporangiospore formation in A. missouriensis. Furthermore, the fact that GsmA orthologues are conserved within the genus Actinoplanes but not in streptomycetes indicates that Actinoplanes has developed an original strategy for the spore maturation in a specific environment, that is, inside a sporangium.

Exposure ofStaphylococcus aureusto Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis

ABSTRACTPeptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptibleStaphylococcus aureusstrain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genesatl,lytM,lytN, andsle1. Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.

Sodium Trimetaphosphate as a Novel Strategy for Matrix Metalloproteinase Inhibition and Dentin Remineralization

The effect of sodium trimetaphosphate (STMP) as an antiproteolytic and remineralizing agent on demineralized dentin was evaluated in vitro. The inhibitory potential of STMP at 0.5, 1.5, 3.5, and 5% against recombinant matrix metalloproteinases (MMPs) MMPs-2 and -9 was assessed by zymography. To investigate its remineralization potential, 40 bovine root specimens were obtained and subjected to a demineralization protocol to produce caries-like dentin lesions. After that, dentin surfaces were divided into 3 areas: (1) mineralized (no treatment); (2) demineralized; and (3) demineralized/treated with STMP and submitted to a pH-cycling associated or not with STMP (1.5, 3.5, or 5% STMP, 10 min of treatment). After that, superficial hardness (SH) and cross-sectional hardness (CSH) were determined. Polarized light microscopy (PLM) was used to qualitatively evaluate mineralization within the caries-like lesions. The zymographic analysis showed that STMP solution is a potent inhibitor of the gelatinolytic activity of MMPs-2 and -9 depending on the dose, since the lowest concentration (0.5%) partially inhibited the enzyme activity, while the higher concentrations completely inhibited enzyme activity. Regarding remineralization effect, only 1.5% STMP solution enhanced both the SH and CSH. PLM showed that the area treated with 1.5% STMP presented similar birefringence as mineralized sound dentin. In conclusion, 1.5% STMP solution is effective as an antiproteolytic agent against MMPs and promotes dentin remineralization.