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81.
MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.  相似文献   
82.
Cell-based approaches offer a potential therapeutic strategy for appropriate bone manufacturing. Capable of differentiating into multiple cell types especially osteoblasts spontaneously, unrestricted somatic stem cell (USSC) seems to be a suitable candidate. Recent studies have shown the involvement of microRNAs in several biological processes. miRNA microarray profiling was applied in order to identify the osteo-specific miRNA signature. Prior to this analysis, osteogenic commitment of osteoblasts was evaluated by measuring ALPase activity, biomineralization, specific staining and evaluation of some main osteogenic marker genes. To support our findings, various in silico explorations (for both putative targets and signaling pathways) and empirical analyses (miRNA transfections followed by qPCR of osteogenic indicators and ALPase activity measurement) were carried out. The function of GSK-3b inhibitor was also studied to investigate the role of WNT in osteogenesis. Transient modulation of multiple osteo-miRs (such as mir-199b, 1274a, 30b) with common targets (such as BMPR, TCFs, SMADs) as mediators of osteogenic pathways including cell-cell interactions, WNT and TGF-beta pathways, suggests a mechanism for rapid induction of the osteogenesis as an anti-miRNA therapy. The results of this research have identified the miRNA signature which regulates the osteogenesis mechanism in USSC. To conclude, our study reveals more details about the allocation of USSCs into osteogenic lineage through modulatory effect of miRNAs on targets and pathways required for creating a tissue-specific phenotype and may aid in future clinical interventions.  相似文献   
83.
Exposure to heavy metals has been associated to a higher incidence of multiple sclerosis. In this work, we present a possible relationship between serum mercury levels and development of multiple sclerosis in Isfahan, the third largest city in Iran. Seventy-four patients affected by multiple sclerosis were retrieved from multiple sclerosis (MS) clinic in Isfahan, Iran. By matching sex and age, 74 healthy volunteers were chosen as control group. Blood samples were collected and serum mercury content was determined. Serum mercury level in MS patients was significantly higher than controls (9.6 ± 10.17 vs. 5.7 ± 8.6, P = 0.037). Concerning all MS patients, serum mercury value was significantly higher than the mercury concentration founded in control subjects {odd ratio: 2.39 (CI, 1.96–2.94), P = 0.00}. Serum mercury level is higher in MS patients with odd ratio equal to 2.39 compared with healthy individuals. It may reveal that high mercury levels in serum might help MS development in susceptible individuals. More studies with larger sample size are needed to confirm this hypothesis.  相似文献   
84.
The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (~50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (~90%). Moreover dependency of hMSCs to size of transfected nucleic acid and time-dependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, which eventually improves nonviral cell-mediated gene therapy.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9430-9) contains supplementary material, which is available to authorized users.  相似文献   
85.
More recently, we have reported the beneficial effects of ghrelin in improvement of histopathological features of the rat testis following local heat exposure. However, the exact mechanism and the precise role of apoptosis- and proliferation-specific proteins in this regeneration process remained to be explored. Thus, thirty adult male Wistar rats were allotted for the experiment and subdivided equally into three groups: control-saline (CS), heat-saline (HS) and heat-ghrelin (HG). The scrota of HS and HG groups were immersed once in water bath at 43 °C for 15 min. HG animals received 2 nmol of ghrelin subcutaneously immediately after heating every other day until day 60 and the other groups were given physiological saline using the same method. The testes of all groups were taken after rat killing on days 30 and 60 after heat treatment for immunocytochemical detection of pro-apoptotic factor Bax, anti-apoptotic protein Bcl-2 and proliferation-associated peptide PCNA in the germ cells. Ghrelin could significantly suppress the Bax expression in spermatocytes compared to the HS group at day 30 (P < 0.05). Likewise, the mean percentages of spermatogonia containing Bax substance were lower in ghrelin-exposed animals, however the differences were not statistically significant. There were immunoreactive cells against Bcl-2 in each germ cell neither in the control nor in the heated animals of experimental groups. In contrast, the number of PCNA immunolabeling cells were higher in HG group in compared to HS or CS animals on both experimental days (P < 0.001). Down-regulation of Bax expression concurrent with overexpression of PCNA in HG group indicates the ability of ghrelin in acceleration of testicular germ cells regeneration following heat stress. These findings indicate that ghrelin may be used as a novel and efficient antioxidant agent to induce resumption of spermatogenesis upon environmental heat exposure.  相似文献   
86.
The brain mechanism of extracting visual features for recognizing various objects has consistently been a controversial issue in computational models of object recognition. To extract visual features, we introduce a new, biologically motivated model for facial categorization, which is an extension of the Hubel and Wiesel simple-to-complex cell hierarchy. To address the synaptic stability versus plasticity dilemma, we apply the Adaptive Resonance Theory (ART) for extracting informative intermediate level visual features during the learning process, which also makes this model stable against the destruction of previously learned information while learning new information. Such a mechanism has been suggested to be embedded within known laminar microcircuits of the cerebral cortex. To reveal the strength of the proposed visual feature learning mechanism, we show that when we use this mechanism in the training process of a well-known biologically motivated object recognition model (the HMAX model), it performs better than the HMAX model in face/non-face classification tasks. Furthermore, we demonstrate that our proposed mechanism is capable of following similar trends in performance as humans in a psychophysical experiment using a face versus non-face rapid categorization task.  相似文献   
87.
Oxidative stress is one of the factors associated with decline in fertility and betaine has been shown to bear antioxidant and methyl donor properties in our recent studies. Thus, we designed the present study to examine antioxidant and methyl donor abilities of betaine in oxidative stress induced by ethanol in the rat testes. The adult male Sprague-Dawley rats were divided into four experimental groups and treated daily for 2?months as follows: control, ethanol (4?g/kg, orally), betaine (1.5?% of total diet, orally), and betaine plus ethanol (betaine, 1.5?% of total diet and after 120?min, ethanol 4?g/kg). Sperm motility and concentration significantly increased in betaine group when compared to the ethanol?Ctreated rats. The main antioxidant enzyme (GPx) activity significantly increased (in order compensatory) in ethanol-treated rats when compared to betaine group while, antiperoxidative enzyme (CAT) activity significantly increased in betaine plus ethanol group as compared to ethanol-treated rats. Total homocysteine (tHcy) and TBARS concentration (as a lipid peroxidation marker) also significantly decreased in betaine and betaine plus ethanol groups as compared to ethanol-treated rats. Overall, methyl donor and antioxidant properties of betaine are promising and reduce the elevated tHcy and TBARS concentrations in betaine plus ethanol group. Therefore, betaine might be used as a potential therapy in hyperhomocysteinemia and oxidative stress induced by ethanol in alcoholism.  相似文献   
88.
Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40-50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly beta- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.  相似文献   
89.

Background

Automated image analysis, measurements of virtual slides, and open access electronic measurement user systems require standardized image quality assessment in tissue-based diagnosis.

Aims

To describe the theoretical background and the practical experiences in automated image quality estimation of colour images acquired from histological slides.

Theory, material and measurements

Digital images acquired from histological slides should present with textures and objects that permit automated image information analysis. The quality of digitized images can be estimated by spatial independent and local filter operations that investigate in homogenous brightness, low peak to noise ratio (full range of available grey values), maximum gradients, equalized grey value distribution, and existence of grey value thresholds. Transformation of the red-green-blue (RGB) space into the hue-saturation-intensity (HSI) space permits the detection of colour and intensity maxima/minima. The feature distance of the original image to its standardized counterpart is an appropriate measure to quantify the actual image quality. These measures have been applied to a series of H&;E stained, fluorescent (DAPI, Texas Red, FITC), and immunohistochemically stained (PAP, DAB) slides. More than 5,000 slides have been measured and partly analyzed in a time series.

Results

Analysis of H&;E stained slides revealed low shading corrections (10%) and moderate grey value standardization (10 – 20%) in the majority of cases. Immunohistochemically stained slides displayed greater shading and grey value correction. Fluorescent stained slides are often revealed to high brightness. Images requiring only low standardization corrections possess at least 5 different statistically significant thresholds, which are useful for object segmentation. Fluorescent images of good quality only posses one singular intensity maximum in contrast to good images obtained from H&;E stained slides that present with 2 – 3 intensity maxima.

Conclusion

Evaluation of image quality and creation of formally standardized images should be performed prior to automatic analysis of digital images acquired from histological slides. Spatial dependent and local filter operations as well as analysis of the RGB and HSI spaces are appropriate methods to reproduce evaluated formal image quality.
  相似文献   
90.
CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.  相似文献   
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