Congenitalcholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenitalandacquiredcholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogene- sis of the abnormality. However, the exact pathogenic mechanisms by which cholesteato- mas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cho- lesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only incongenital cho- lesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Proteinexpression was con- firmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar proteinexpression patterns and that 127 spots were exclusively expressed incongenitalcholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homo- log (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analy- sis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed incongenital cholesteatoma matrices, but not inacquiredcholesteatomas. Our study shows that proteinexpression patterns are completely different incongenitalcholesteatomas, acquiredcholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed incongenital cholesteatoma tissue. Our data indi- cates that congenital cholesteatoma origins may differ from those of acquired cholesteato- mas, which originate from retraction pocket epithelia.
Protein spots with differentialexpression were manually excised from Coomassie Blue-Stained 2D gels using a sterile scalpel and digested by sequencing grade, modified porcine trypsin (Promega, Madison, WI). Samples were applied directly to the MALDI target plate and positive- ion MALDI mass spectra were obtained using an Applied Biosystems 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) in reflectron mode. MS spectra were acquired over a mass range of m/z 800-4000. Final mass spectra were internally calibrated using the tryptic autoproteolysis products at m/z 842.509 and 2211.104. Monoisotopic masses were obtained from centroids of raw, unsmoothed data. The twenty strongest peaks with a signal to noise greater than 50 were selected for CID-MS/MS analysis.
exponential or stationary phase under different polymer accumu- lation conditions, using sodium octanoate or glucose as carbon sources. The composition of the proteins bound to the PHA granules was also analyzed from cells subjected to 5h of carbon starvation. SDS-PAGE of PHA granules, extracted from cells grown under different conditions, displayed a similar qualitative pattern of proteins (data not shown). For that reason, only the proteins obtained from granules extracted from 24 h-cultures, grown with sodium octanoate, were identified. Based on the molecular weight of the proteins normally found in PHA granules, and their relative amount in the SDS-PAGE (Fig. 3), nine bands were excised for the gel and subsequent identified by peptide fingerprint analysis (Table 4). Several proteins directly involved in PHA metabolism, such as the synthases PhbC and PhaC1, the phasin like protein PhaI and the phasin like protein PhbP, were identified among the granule bound proteins. PhbP represented the major band (Fig 3). In addition, PhbX, encoded by an ORF located downstream of phbP, was also found in the granules. Another four proteins, not known to be related to PHA metabolism, were also found: the cytosolic aminopeptidase PepA, a possible secretion protein, the heat shock protein Hsp20 and the DNA binding protein HU (Table 4).
Twenty-five µg of protein was separated on a 10% SDS polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. Membranes were blocked with 5% non fat dry milk in TBS containing 0.05% Tween 20 and were incubated overnight with one of the following antibodies: anti- β-actin (Abcam, Cambridge, UK) at 1:2500; anti- DNM1L (Abcam, Cambridge, UK) at 1:500; anti-OPA1 (Abcam, Cambridge, UK) at 1:2000; anti-synaptophysin (Abcam, Cambridge, UK) at 1:1500; and anti-caspase 3 (Abcam, Cambridge, UK) at 1:1000. Goat anti-mouse IgG and goat polyclonal anti-rabbit IgG (both from Abcam, Cambridge, UK) secondary antibodies were used and detected using ECL Western Blotting Substrate Kit (Abcam, Cambridge, UK). Pre-stained molecular weight protein markers (SuperSignal Molecular Weight Protein Ladder, Thermo Scientific, Rockford, USA) were used to determine the detected bands’ molecular weight and confirm target specificity of antibodies. The densitometry quantification was performed using ImageJ software (http://rsb.info.nih.gov/ij/). Total blotting protein levels of samples were normalized according to each sample’s β-actin protein levels [adapted from 36].
TRIC assembly at tTJ in cochlear and vestibular epithelial cells appears to play a key role in hearing, as mutations in the TRIC gene were associated with deafness (Riazuddin et al., 2006). It is known that TRIC protein is present at tricellular contacts of vestibular and cochlear epithelia and mutations of TRIC-a, the longest human isoform, have been reported to result in a human hereditary disease, nonsyndromic deafness (DFNB49). A common feature of the four DFNB49 TRIC alleles is that they encode predicted truncated proteins that lack the ability to bind to the scaffolding protein ZO-1, because of the loss of the conserved C-terminal occludin-ELL domain, and as previously described, this can interfere with the localization to TJs. In contrast, Raleigh, et al., (2010) suggested that TRIC may interact with ZO-1 at another site, because their studies show that C-terminus of TRIC was not able to bind ZO-1 GK domain. Alternatively, minor differences between the TRIC tails constructs used in both studies may explain the disparate observations. In fact, it has been speculated the relation between mutations in TJ gene and deafness. Mutations in claudin 14 gene are associated with different hearing thresholds, including profound andcongenital deafness, but the role of this proteinin hearing process is still unknown (Bashir et al., 2010; Ben-yosef et al., 2003). Yet, the only obvious phenotype of mutant alleles of Tric is deafness. It is possible that some other molecule compensates for the absence of wild-type TRIC in other epithelial cell types but not in the inner ear.
The patient is a full-term neonate, born from e Cesarean section, presenting sings of distress and meconium aspiration (Apgar 6, 8 and 9). Right after birth the patient showed signs of respiratory discomfort and meconium aspiration, and was intubated. Chest X-rays, however, ruled out such diagnostic possibil- ity and the patient was extubated. The neo- nate still presented stridor during inhalation and showed sings of discomfort in times of physical strain, mainly as he cried and was fed. Nasal endoscopy showed a cyst growing towards the aryepiglottic fold consistent with a saccular cyst.
In fact, in the cagA + cases, nuclear positivity of MYC was more associated with the diffuse subtype (63%), although with no statistical difference, while the negativity was significantly associated with the intestinal subtype (83%). Previously, our research group showed the relevance of MYC in diffuse type tumors where it was associated with p27 negativity . Since the histological subtypes act through different carcinogenic pathways, our findings indicate that in a relevant number of cases of the intestinal tumors, the carcinogenesis process H. pylori-related does not occur through the MYC proteinexpression. Interest- ingly, the observation that most of the few cytoplasmic MYC positive cases were in the intestinal subtype and were associated with the presence of cagA + (8/12, 66.7%), suggest that in these few tumors there is a blockage in the transport of MYC protein through the nuclear membrane. Additional studies of the molecular events (transcriptional or post-transcriptional level) are required to better under- stand this and other molecular markers involved in the gastric cancer carcinogenesis process considering the H. pylori and cagA gene.
Microgravity has significant effects on numerous microbial characteristics . And studies concerning the influences of microgravity on microbial cells are drawing much attention because such information will lead to the advancement of knowledge and application about space biotechnology . However, SMG was more applicable and feasible to most researchers due to much lower cost, without any further need of spaceflight and extreme sensitive instrumentations, which defi- nitely make the experiments out of range for future projects. Therefore a series of ground-based suspension culture bioreactors which could model various aspects of spaceflight were invented . One such bioreactor named as high-aspect-ratio vessel (HARV, Synthecon)  was used in this study. The HARV employed for cell suspension culture and tissue growth permits cell growth in suspension and minimizes the fluid shear levels encountered by cells [1,4]. When the cell culture vessel rotates, microbial cells do not settle down but revolve around a horizontal axis and continuously fall through the fluid at 16g terminal velocity condition, which lead to 1,2610 22 gravity environment . Exchange of dissolved gas has been achieved through a permeable membrane at the back of this vessel.
Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb – girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, a-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.
Although the molecular mechanism by which GCs influence CBG levels is unclear, the Cbg promoter has been shown to be transcriptionally regulated via the GR [30–32]. However, while the Cbg promoter is modulated by GCs [30,32,33], no glucocor- ticoid response elements (GREs) seem to be present in the mouse, rat or human CBG gene proximal promoters [25,28], suggesting tethering of the GR to other transcription factors rather than binding directly to DNA. Footprints P3–P5 in the Cbg promoter resemble recognition sequences for DBP, HNF3a and C/EBPb or NF-IL6 , and, although binding of these transcription factors has not been confirmed, it is interesting to note that HNF3a and C/EBPb have been reported to be important in GR-mediated signaling [34,35]. Specifically, protein-protein interaction of the GR with C/EBPb has been reported  and both C/EBPb and HNF3a are considered pioneer transcription factors as they increase chromatin accessibility at GC-responsive genes, thereby facilitating GR interaction with GC-responsive promoters [36,37]. In this study, the molecular mechanism of action of GC- mediated repression of CBG expression was investigated. Because no consensus GR binding sites have been identified within the CBG gene promoter, delineation of GR responsiveness within the proximal rat CBG gene promoter was performed. This work identified the region between 2295 and 2145bp 59 of the transcription start, which contains putative binding sites for C/ EBPb, HNF3a and DBP, as being important for GC responsive-
with oscillatory expression are known inhibitors of the pathways that induce their expression. Such simple feedback mechanisms can generate oscillations, provided the presence of a delay in the process (Aulehla and Pourquie, 2008). Dale, et, al (2003) first identified a negative feedback loop acting in the chick PSM, in which lfng (Lunatic fringe is a Notch target gene encoding glycosyltransferase that modulates Notch signalling will act, in turn, to inhibit Notch signalling and thus regulate lfng’s own expression. In this model, the NICD domain, after translocated to the nucleus, will activate hairy1, hairy2 and lfng genes. Lnfg protein modifies Notch, which becomes less sensitive to activation by Delta , yet it’s a transient and periodic effect due to the short life and rapid turnover of the Lfng protein. Oscillations are thus generated by alternation between activation of lfng expressionand repression of Notch by Lfng. The influence of NICD on lfng exerted via hairy genes will be delayed relative to the influence exerted directly, and this phase shift can, in principle, allows the two influences to act in synergy (Giudicelli and Lewis, 2004).This study proposed that this negative feedback loop involving these genes represents a core component of the avian segmentation clock mechanism (Giudicelli and Lewis, 2004) (Figure 1.3).
CASE REPORT: A 19-year-old woman with two gesta- tions and one parity (G2P1) had exhibited deep venous thrombosis in her previous puerperal pe- riod. Investigation of thrombophilic factors revealed ACA-IgM and heterozygous C677T mutation in the MTHFR gene. Lupus anticoagulant, protein C, protein S and antithrombin III deficiencies, and Leiden factor V and the G20210A mutation in the prothrombin gene, were not detected. The patient received 55,000 IU of subcutaneous heparin daily, from the 15 th to the 36 th week of pregnancy, when
We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expressionin VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G 0 /G 1 , inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.
originally isolated, particularly the ability to replicate indefinitely. For example, while a recent RNA-seq analysis of HBV-mediated gene expression changes in the Huh7 cell line did identify metabolic pathways as being altered by HBV, only minimal identification of cell cycle-related DEG was reported . This may be due to the altered baseline of cell cycle regulation occur- ring in these transformed cells, limiting the ability to detect alteration to genes whose expres- sion is already drastically altered. Such changes are more easily identified in a primary hepatocyte model, as time-mediated changes which separate cultured primary hepatocytes from freshly isolated cells can be directly identified and serve as a baseline for expression changes, instead of the endogenously altered baseline present in cell lines. In fact, multiple studies have demonstrated the significant phenotypic differences between primary hepatocytes and cell lines [41, 80], which were also recently supported by a transcriptome analysis of the HepG2 hepatoblastoma cell line. This study compared HepG2 cells to primary liver tissue and confirmed that the majority of up-regulated genes were associated with cancer and cellular pro- liferation pathways, prompting the authors to warn against the use of HepG2 cells in liver can- cer-related studies of the transcriptome . On the other hand, while we do identify time- mediated gene expression changes in our PRHs model, these numbers are likely biased by genes with a low expression level or small fold change. This is supported by the high correlation when the complete gene expression profile in cultured PRHs is compared between 0hr samples and later time points. Based on this, and on the data reported here, the use of primary hepato- cytes seems to be an appropriate cell-culture-based model for identifying alteration of cellular pathways, including those altered in the context of HBV infection.
Notes from the foregoing that the centralized and decentralized meet in development management style. But they were taking two different meanings Decentralization means the distribution of powers, and responsibilities involving the administrative bodies, and spatial participation in all matters relating to the development process of local, regional and international dimensions and prevent the authorities and institutions at least the rank limit participation to take decisions and policies related to development and there are those who know the centralization and decentralization in educational administration that the central It means the concentration of power at the top management level, and education in the state
This study investigated the extraction and purification technology of matrine from Sophora flavescens Ait.. After the single factor experiments and the optimization experiment through Box-Behnken central composite design and response surface analysis, the optimal extraction conditions were obtained. On addition, through the acidification and alkalization treatment, the impurities in the crude matrine product were removed. After ion exchange resin adsorption, the final matrine product was obtained, which had a purity of 81.56%. This indicate that, the extraction and purification technology of matrine from Sophora flavescens Ait. developed in this study is relatively satisfactory. Next, the inhibitory effects of matrine on human NPC CNE-2 cells were evaluated. Results showed that, matrine could obtain inhibit the growth of CNE-2 cells, promote the cell apoptosis, and arrest most CNE-2 cells in G0/G1 phase. This indicates that, matrine has inhibitory effects of CNE-2 cells.
that the one having more TCRs would down-regulate a greater number, with this number being correlated with the magnitude of the signal transduced (Schrum et al., 2000). A sim- ilar point had been made in an early work using human T cell clones, which argued for the number of TCR triggered upon stimulation as a threshold for the acquisition of certain effector functions (Viola and Lanzavecchia, 1996). Some of the clones in that study (Vi- ola and Lanzavecchia, 1996) had multiple β chains, and multiple TCRs clonotypes, each expressed in different amounts when compared among clones. Indeed, cells that have two functional α and/or β chains may have multiple αβ pairings, and hence multiple TCRs clonotypes. They may be obtained, for instance, by crossing two TCR-transgenic mouse strains. The levels of each TCR clonotype in the cells expressing many of them may be lower than the levels in cells expressing only one clonotype, for example in the case that the total number of TCRs does not vary considerably in these two groups of cells. Indeed, such reduced levels of each of multiple clonotypes have been reported by some studies, which also found evidence for a correlation between the expression level of a particular clonotype and the response elicited upon stimulation of that clonotype (Dave et al., 1999; Legrand and Freitas, 2001a,b; Schrum and Turka, 2002). There are, however, two potentially com- plicating factors in these data. First, some particular αβ pairs tend to be expressed at higher levels than other pairs in the same cells. This results in levels of a particular TCR clono- type that are much lower (for example, 10-fold; Dave et al., 1999) than the levels in cells having that particular clonotype alone, such that the differences that are necessary in order for functional consequences to be observed may be relatively large. Second, the fact that, upon stimulation with a given peptide, expected to engage only a given TCR clonotype, that peptide may interact with some of the additional clonotypes present in cells and pro- vide inhibitory signals (similar to what is known as TCR antagonism; see, for example, Yang and Grey, 2003).
T here are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS). However, the true origin and the phenotype of the DLS are not known. Objective:To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands. Material and Methods: Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19) and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS) and Alcian Blue. In each captured microscopic ield, the DLS were counted to establish a percentage for the staining proile. The statistical analysis was accomplished using Student’s t-test, the Mann-Whitney test and Pearson’s correlation coeficient (p<0.05). Results: Comparing both groups, only CK 19 showed a statistically signiicant difference (p=0.033), with the strongest expressionin the elderly group. There was no signiicant difference between PAS and Alcian Blue (p=0.270). In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001), but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315). There was no immunostaining for Ki-67. Conclusions: DLS demonstrate a ductal phenotypic proile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia.
Immunoprecipitation (ChIP) across MYC with anti-C-terminal Brd4 antibody. Each experiment was performed twice, analyzed in triplicate via real-time PCR and reported as the mean and standard deviation of the two experiments. A representation of the promoter area of MYC is provided for orientation. (B) Western blotting to detect (far left) Brd4 (~180 KD) and (middle) Brd4-S484/488-phos (P-Brd4, ~220 KD) was performed three times. A non-specific band detected with phopsho-Brd4 antibody is denoted with an asterisk. Typical results are shown with densitometry analysis relative to CREB expression, which is used as a normalization control (far right).
Tolerance to P deficiency may be reached by increasing the P uptake capacity. However, at low-P conditions, P contents reflect not only the plants ability to extract P from soils, but also their capacity of P remobilization and redistribution during the crop cycle. Phosphorus uptake capacity depends on several plant characteristics; one of them is the root density. The increase in root system superficial area increases the plant’s ability to access and uptake P. Wiethölter (2004) suggests that the 'Toropi' genotype has a higher capacity of P extraction due to an extensive root system. No significant differences were observed, neither in root dry matter (Table 1) nor in root length, between 'Toropi' and 'Anahuac' growing in low-P in hydroponics, at least at the initial growth stages. However, it was possible to visualize differences in the tolerance of low-P conditions between genotypes, especially in the color of the shoot. Similar observations were described by Silva et al. (2008). The observed differences between genotypes may be related to different crop cycles: 'Anahuac' is classified as an early cycle variety, and 'Toropi', as a late cycle variety (Cunha et al., 1997). Some traits usually related to low-P tolerance, such as morphological root alterations, mycorrhyzae association and enzyme secretion capacity, are not pertinent in hydroponics studies, since the P in the solution is in the inorganic form and promptly available.