Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.

CA15-3 and alkaline phosphatase as predictors for breast cancer recurrence: a combined analysis of seven International Breast Cancer Study Group trials

BACKGROUND: We evaluated the ability of CA15-3 and alkaline phosphatase (ALP) to predict breast cancer recurrence. PATIENTS AND METHODS: Data from seven International Breast Cancer Study Group trials were combined. The primary end point was relapse-free survival (RFS) (time from randomization to first breast cancer recurrence), and analyses included 3953 patients with one or more CA15-3 and ALP measurement during their RFS period. CA15-3 was considered abnormal if >30 U/ml or >50% higher than the first value recorded; ALP was recorded as normal, abnormal, or equivocal. Cox proportional hazards models with a time-varying indicator for abnormal CA15-3 and/or ALP were utilized. RESULTS: Overall, 784 patients (20%) had a recurrence, before which 274 (35%) had one or more abnormal CA15-3 and 35 (4%) had one or more abnormal ALP. Risk of recurrence increased by 30% for patients with abnormal CA15-3 [hazard ratio (HR) = 1.30; P = 0.0005], and by 4% for those with abnormal ALP (HR = 1.04; P = 0.82). Recurrence risk was greatest for patients with either (HR = 2.40; P < 0.0001) and with both (HR = 4.69; P < 0.0001) biomarkers abnormal. ALP better predicted liver recurrence. CONCLUSIONS: CA15-3 was better able to predict breast cancer recurrence than ALP, but use of both biomarkers together provided a better early indicator of recurrence. Whether routine use of these biomarkers improves overall survival remains an open question.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Digital processing and data compilation approach for using remotely sensed imagery to identify geological lineaments in hard-rock terrains : an application for groundwater explorations in Nicaragua

Sustainable yields from water wells in hard-rock aquifers are achieved when the well bore intersects fracture networks. Fracture networks are often not readily discernable at the surface. Lineament analysis using remotely sensed satellite imagery has been employed to identify surface expressions of fracturing, and a variety of image-analysis techniques have been successfully applied in “ideal” settings. An ideal setting for lineament detection is where the influences of human development, vegetation, and climatic situations are minimal and hydrogeological conditions and geologic structure are known. There is not yet a well-accepted protocol for mapping lineaments nor have different approaches been compared in non-ideal settings. A new approach for image-processing/synthesis was developed to identify successful satellite imagery types for lineament analysis in non-ideal terrain. Four satellite sensors (ASTER, Landsat7 ETM+, QuickBird, RADARSAT-1) and a digital elevation model were evaluated for lineament analysis in Boaco, Nicaragua, where the landscape is subject to varied vegetative cover, a plethora of anthropogenic features, and frequent cloud cover that limit the availability of optical satellite data. A variety of digital image processing techniques were employed and lineament interpretations were performed to obtain 12 complementary image products that were evaluated subjectively to identify lineaments. The 12 lineament interpretations were synthesized to create a raster image of lineament zone coincidence that shows the level of agreement among the 12 interpretations. A composite lineament interpretation was made using the coincidence raster to restrict lineament observations to areas where multiple interpretations (at least 4) agree. Nine of the 11 previously mapped faults were identified from the coincidence raster. An additional 26 lineaments were identified from the coincidence raster, and the locations of 10 were confirmed by field observation. Four manual pumping tests suggest that well productivity is higher for wells proximal to lineament features. Interpretations from RADARSAT-1 products were superior to interpretations from other sensor products, suggesting that quality lineament interpretation in this region requires anthropogenic features to be minimized and topographic expressions to be maximized. The approach developed in this study has the potential to improve siting wells in non-ideal regions.

Enhancement of heterologous expression of alkaline phytase in Pichia pastors

Phytic acid is the major storage form of phosphorus and inositol in seeds and legumes. It forms insoluble phytate salts by chelating with positively charged mineral ions. Non-ruminant animals are not able to digest phytate due to the lack of phytases in their GI tracks, thus the undigested phytate is excreted leading to environmental contamination. Supplementation with phytases in animal feed has proven to be an effective strategy to alleviate nutritional and environmental issues. The unique catalytic and thermal stability properties of alkaline phytase from lily pollen (LlALP) suggest that it has the potential to be useful as a feed supplement. Our goal is to develop a method for the production of substantial amounts of rLlALP for animal feed and structural studies. rLlALP2 has been successfully expressed in the yeast, Pichia pastoris. However, expression yield was modest (8-10 mg/L). Gene copy number has been identified as an important parameter in enhancing protein yields. Multicopy clones were selected using Zeocin-resistance-based vectors and challenging transformants to high Zeocin levels under different conditions. Data indicate that increasing selection pressure led to the generation of clones with amplification of both rLlAlp2 and Zeor genes and the two genes were not equally amplified. Additionally, clones generated by step-wise methods led to clones with greater amplification. The effects of transgene copy number and gene sequence optimization on expression levels of rLlALP2 were examined. The data indicate that increasing the copy number of rLlAlp2 in transformed clones was detrimental to expression level. The use of a sequence-optimized rLlAlp2 (op-rLlAlp2) increased expression yield of the active enzyme by 25-50%, suggesting that transcription and translation efficiency are not major bottlenecks in the production of rLlALP2. Lowering induction temperature to 20 oC led to an increase in enzyme activity of 1.2 to 20-fold, suggesting that protein folding or post-translational processes may be limiting factors for rLlALP2 production. Cumulatively, optimization of copy number, gene sequence optimization and reduced temperature led to increase of rLlALP2 enzyme activity by three-fold (25-30 mg/L). In an effort to simplify the purification process of rLlALP2, extracellular expression of phytase was investigated. Extracellular expression is dependent on the presence of an appropriate secretion signal upstream of the transgene native signal peptide(s) present in the transgene may also influence secretion efficiency. The data suggest that deletion of both N- and C-terminal signal peptides of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion of LlALP2 by four-fold. The secretion signal peptide of chicken egg white lysozyme was ineffective in secretion rLlALP2 in P. pastoris. To enhance rLlALP2 secretion, effectiveness of the strong inducible promoter (PAOX1) was compared with the constitutive promoter (PGAP). The intracellular yield of rLlALP2 was about four-fold greater under the control of PGAP compared to PAOX1 and extracellular expression level of rLlALP2 was around eight-fold (75-100 mg/L) greater. The successful production of active rLlALP2 in P. pastoris will allow us to conduct the animal feed supplementation studies and structural studies.

Tenofovir use is associated with an increase in serum alkaline phosphatase in the Swiss HIV Cohort Study

BACKGROUND: Tenofovir (TDF) use has been associated with proximal renal tubulopathy, reduced calculated glomerular filtration rates (cGFR) and losses in bone mineral density. Bone resorption could result in a compensatory osteoblast activation indicated by an increase in serum alkaline phosphatase (sAP). A few small studies have reported a positive correlation between renal phosphate losses, increased bone turnover and sAP. METHODS: We analysed sAP dynamics in patients initiating (n = 657), reinitiating (n = 361) and discontinuing (n = 73) combined antiretroviral therapy with and without TDF and assessed correlations with clinical and epidemiological parameters. RESULTS: TDF use was associated with a significant increase of sAP from a median of 74 U/I (interquartile range 60-98) to a plateau of 99 U/I (82-123) after 6 months (P < 0.0001), with a prompt return to baseline upon TDF discontinuation. No change occurred in TDF-sparing regimes. Univariable and multivariable linear regression analyses revealed a positive correlation between sAP and TDF use (P < or = 0.003), but no correlation with baseline cGFR, TDF-related cGFR reduction, changes in serum alanine aminotransferase (sALT) or active hepatitis C. CONCLUSIONS: We document a highly significant association between TDF use and increased sAP in a large observational cohort. The lack of correlation between TDF use and sALT suggests that the increase in sAP is because of the bone isoenzyme and indicates stimulated bone turnover. This finding, together with published data on TDF-related renal phosphate losses, this finding raises concerns that TDF use could result in osteomalacia with a loss in bone mineral density at least in a subset of patients. This potentially severe long-term toxicity should be addressed in future studies.

Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways

Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.