Identification of cytoplasmic and membrane-associated complexes in human embryonic stem cells using blue native PAGE

Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.     

Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.     

Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.     

Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae)

The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases.     

Chaperone-like activity of α-cyclodextrin via hydrophobic nanocavity to protect native structure of ADH

The chaperone action of α-cyclodextrin (α-CyD), based on providing beneficial microenvironment of hydrophobic nanocavity to form molecular complex with alcohol dehydrogenase (ADH) was examined by experimental and computational techniques. The results of UV-vis and dynamic light scattering (DLS) indicated that the chaperone-like activity of α-CyD depends on molecular complex formation between α-CyD and ADH, which caused to decrease the amount and size of polymerized molecules. Computational calculations of molecular dynamic (MD) simulations and blind docking (BD) demonstrated that α-CyD acts as an artificial chaperone because of its high affinity to the region of ADH’s two chains interface. The hydrophobic nanocavity of α-CyD has the ability to form inclusion complex due to the presence of phenyl ring of aromatic phenylalanine (Phe) residue in the dimeric intersection area. Delocalization of ADH subunits, which causes the exposure of Phe110, takes part in the enzyme polymerization and has proven to be beneficial for aggregation inhibition and solubility enhancement within the host α-CyD-nanocavity.     

Expression profile of IL-8 and growth factors in breast cancer cells and adipose-derived stem cells (ASCs) isolated from breast carcinoma

Adipose-derived stem cells (ASCs) are regarded as a major player of breast cancer microenvironment. By production of various growth factors and expression of regulatory molecules, it is postulated that ASCs protect breast cancer cells from the host immune responses. In this study, the expressions of insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), CXCL8 (IL-8) in breast cancer cells and adipose-derived stem cells isolated from breast tissue of women with breast cancer were investigated. The results were analyzed comparatively in normal ASCs isolated from healthy normal women. In case of breast cancer tissues, results were analyzed between high stage and low stage patients. The expressions of extracted mRNAs were determined using real-time quantitative RT-PCR. As a result, in breast cancer tissues, IGF-1 and IL-8 mRNAs had 28.6 and 56-fold more expressions in high stage compared to low stage patients. In ASCs, relative quantifications (RQ) of VEGF, IL-8, HGF and IGF-1 was about 2-fold higher in patients than controls. Data of this study conclude that presence of resident ASCs within the scaffold of breast tissue may support breast tumor growth and progression through the expressions of tumor promoting factors.     

Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-alpha in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-alpha; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-alpha antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-alpha groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-alpha. Hepatic TNF-alpha and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-alpha compared with those sham injected. In addition, ileal luminal levels of both TNF-alpha and IL-18 were significantly decreased in the anti-TNF-alpha-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-alpha group. These data show that hepatic TNF-alpha is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-alpha could be used as a potential therapy for human NEC.     

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.     

Relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth in menopausal women

doi: 10.1111/j.1741‐2358.2010.00403.x Relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth in menopausal women Objectives: To evaluate the relationship of stimulated whole saliva cortisol level with the severity of a feeling of dry mouth (DM) in menopausal women. Background: A feel of DM is a major complaint for many elderly individuals and strongly associated with the menopause. The exact mechanisms that mediate sensation of DM in menopausal women have not been firmly established. Methods: A case–control study was carried out on 104 selected menopausal women with/without a feeling of DM, conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. Xerostomia Inventory (XI) score was used as an index of DM severity. Stimulated whole saliva cortisol concentration (stimulated by chewing standard‐sized paraffin for 60 s) was measured by ELISA. Statistical analysis by Student’s t‐test and Spearman correlation was used. Results: The mean cortisol concentration of saliva, but not saliva cortisol output, was significantly higher in the cases than in the controls. There was significant positive correlation between XI score and concentration (r = 0.357, p = 0.000) or output (r = 0.223, p = 0.017) of stimulated whole saliva cortisol. Conclusions: It appears that stimulated whole saliva cortisol is high in menopausal women with a feeling of DM.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.