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1.
The applications of Raman microspectroscopy have been extended in recent years into the field of clinical medicine, and specifically in cancer research, as a non‐invasive diagnostic method in vivo and ex vivo, and the field of pharmaceutical development as a label‐free predictive technique for new drug mechanisms of action in vitro. To further illustrate its potential for such applications, it is important to establish its capability to fingerprint drug mechanisms of action and different cellular reactions. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 48 and 72 hours exposure of lung cancer cell lines, A549 and Calu‐1, after exposure to Actinomycin D (ACT) and Raman micro‐spectroscopy was used to track its mechanism of action at subcellular level and subsequent cellular responses. Multivariate data analysis was used to elucidate the spectroscopic signatures associated with ACT chemical binding and cellular resistances. Results show that the ACT uptake and mechanism of action are similar in the 2 cell lines, while A549 cells exhibits spectral signatures of resistance to apoptosis related to its higher chemoresistance to the anticancer drug ACT. The observations are discussed in comparison to previous studies of the similar anthracyclic chemotherapeutic agent Doxorubicin. A, Preprocessed Raman spectrum of ACT stock solution dissolved in sterile water and mean spectrum with SD of (B) nucleolus, (C) nucleus and (D) cytoplasm of A549 cell lines after 48 hours exposure to the corresponding IC50. 相似文献
2.
Saeidi N Guo X Hutcheon AE Sander EA Bale SS Melotti SA Zieske JD Trinkaus-Randall V Ruberti JW 《Biotechnology and bioengineering》2012,109(10):2683-2698
Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold‐based and scaffold‐free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS—five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long‐term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick‐freeze/deep‐etch electron microscopy. Both the scaffold‐free and the collagen‐based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell‐derived matrix in the scaffold‐based constructs was significantly lower than that produced by scaffold‐free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell‐derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Biotechnol. Bioeng. 2012; 109: 2683–2698. © 2012 Wiley Periodicals, Inc. 相似文献
3.
Krishna CM Sockalingum GD Venteo L Bhat RA Kushtagi P Pluot M Manfait M 《Biopolymers》2005,79(5):269-276
A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging. 相似文献
4.
Wahl JK 《Journal of cellular biochemistry》2005,96(2):390-403
Plakophilins (pkp-1, -2, and -3) comprise a family of armadillo-repeat containing proteins that are found in the desmosomal plaque and in the nucleus. Plakophilin-1 is most highly expressed in the suprabasal layers of the epidermis and loss of plakophilin-1 expression results in skin fragility-ectodermal dysplasia syndrome, which is characterized by a reduction in the number and size of desmosomes in the epithelia of affected individuals. To investigate the role of plakophilin-1 during desmosome formation, we fused plakophilin-1 to the hormone-binding domain of the estrogen receptor to create a fusion protein (plakophilin-1/ER) that can be activated in cell culture by the addition of 4-hydroxytamoxifen. When plakophilin-1/ER was expressed in A431 cells it was incorporated into endogenous desmosomes and did not disrupt desmosome formation. A derivative of A431 cells (A431D) do not form desmosomes, even though they express all the components believed to be necessary for desmosome assembly. Expression and activation of plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining on the cell surface. Co-expression of a classical cadherin (N-cadherin) and plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining at cell-cell borders. These data suggest that plakophilin-1 can induce assembly of desmosomal components in A431D cells in the absence of a classical cadherin; however a classical cadherin (N-cadherin) is required to direct assembly of desmosomes between adjacent cells. The activatable plakophilin-1/ER system provides a unique culture system to study the assembly of the desmosomal plaque in culture. 相似文献
5.
To elucidate factors involved in the oral-aboral axis specification, several observations and experiments were undertaken using the sand dollar Scaphechinus mirabilis. Unlike in Strongylcentrotus purpuratus, localization of mitochondria was not detected in unfertilized eggs. After fertilization, however, the bulk of mitochondria became localized to the opposite side of sperm entry. The first cleavage divided this mitochondrial cluster into daughter blastomeres. On the other hand, a second cleavage produced daughter blastomeres containing quite different amounts of mitochondria. To know whether such mitochondrial localization affects the oral-aboral axis specification, 4-cell-stage embryos were separated along the second cleavage plane. Although both half embryos developed into morphologically normal plutei, some differences, such as the number of pigment cells, were noticed between the siblings. In contrast, cell tracing revealed that the first cleavage separated the oral from the aboral part in most cases, indicating that the unequal distribution of mitochondria is not critical for the oral-aboral axis specification. Further, stained and non-stained half embryo fragments were combined. Such combined embryos developed into normal plutei with a single oral-aboral axis. The plane dividing labeled and non-labeled parts were incident, oblique or perpendicular to the median plane of the combined embryo, and the appearance frequencies of those labeling patterns were similar to those obtained by cell tracing in intact embryos. Interestingly, the half fragments derived from embryos inseminated earlier showed a tendency to form the oral part. These suggest that several factors as well as the localized cytoplasmic components would be involved in the specification process of oral-aboral axis. 相似文献
6.
Surface-enhanced Raman spectroscopy (SERS) in a silver sol assisted by density functional theory (DFT) calculations is shown to be a promising tool in the characterization of platinum complexes and their interaction with nucleic acid bases. This is demonstrated using cisplatin and guanine as a model. The energies and geometric parameters of cisplatin, guanine, and their reaction products are calculated at Becke's nonlocal three parameter exchange and correlation functional and the Lee-Yang-Parr correlation functional level using the 6-31++G(d,p) basis set on the light elements and the effective core potential by Hay and Wadt on platinum. Available X-ray crystallography data are mostly in agreement with predictions within the experimental precision level, although Pt-N bond lengths tend to be systematically overestimated. The normal Raman spectrum of cisplatin is assigned. The SERS spectra of cisplatin and its reaction product with guanine are measured from 10(-6) M aqueous solution. The observed spectral changes in the SERS spectrum of guanine upon cisplatin binding are modeled by DFT calculations. The best agreement between theory and experiment is achieved when the adsorbed reaction product is assumed to be the 1:1 adduct cis-Pt(NH3)2ClG in which Pt is bound to N7 and guanine is deprotonated at N9. 相似文献
7.
Qing Wang Chenchen Zhou Xiaohai Yang Lin Liu Kemin Wang 《Journal of molecular recognition : JMR》2014,27(11):676-682
Because cell‐specific aptamers have high potential for biomedical applications, investigation of the interaction between cell and its aptamers may be of key importance for an improved understanding of biochemical processes. Herein, the interaction between human lung adenocarcinoma A549 cell and its four aptamers was explored using single‐molecule force spectroscopy (SMFS). The values of the unbinding force varied from 117.1 to 171.0 pN at the loading rate of 1.8 × 105 pN/s. Based on the dependence of singe molecule force on the atomic force microscopy loading rate, the corresponding kinetic parameters were obtained. The results revealed two activation barriers and two transient states in the unbinding process of aptamer/cell interaction. More importantly, the binding sites on A549 cells with its four aptamers were defined to be different using SMFS and flow cytometry. This work demonstrated that SMFS can be used as a powerful tool for exploring the aptamer/cell binding behavior at the single‐molecule level, and may provide valuable information for the design and application of aptamer probes. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
8.
Matrix-cytoskeletal interactions in the developing eye 总被引：5，自引：0，他引：5
E D Hay 《Journal of cellular biochemistry》1985,27(2):143-156
The embryonic avian corneal epithelium in vitro responds to extracellular matrix (ECM) molecules in either soluble or polymerized form by flattening its basal surface, organizing the basal cortical actin cytoskeleton, and stepping up its production of corneal stroma twofold. Embryonic corneal epithelia, like hepatocytes and mammary gland cells, seem to contain heparan sulfate proteoglycan (HSPG) in their plasmalemma, which may interact with actin on the one hand or underlying collagen on the other. Work on the corneal epithelium suggests that, in addition to HSPG, specific glycoprotein receptors for laminin and collagen exist in the basal plasmalemma and play the critical role in actually organizing the basal epithelial cytoskeleton. As yet, uncharacterized proteins may link such receptors to actin. We suggest that ECM-dependent organization of the cytoskeleton is responsible for ECM enhancement of corneal epithelial differentiation. Cell shape and exogenous ECM also affect mesenchymal cell differentiation. In the case of the corneal fibroblast migrating in collagen gels, an actin cortex present around the elongate cell seems to interact with myosin in the cytosol to bring about pseudopodial extension. Both microtubules and actin microfilaments are involved in fibroblast elongation in collagen gels. It follows from the rules presented in this review that the mesenchymal cell surface is quite different from the epithelial cell surface in its organization. Nevertheless, epithelial cell surface-ECM interaction can be modified in the embryo at particular times to permit predesignated epithelial-mesenchymal transformations, as for example at the primitive streak. Though basal surfaces of definitive, nonmalignant epithelia adhere rather strictly to the rules of epithelium-ECM interaction and do not invade underlying ECM, the environment can be manipulated in vitro to cause these epithelia to send out pseudopodia and give rise aberrantly to mesenchymal cells in collagen gels. Further study of this phenomenon should cast light on the manner in which epithelial and mesenchymal cells organize receptors for matrix molecules on their cell surfaces and develop appropriate cytoskeletal responses to the extracellular matrix. 相似文献
9.
Ralf Bienert Markus Zeeb Lubomir Dostl Anette Feske Christine Magg Klaas Max Heinz Welfle Jochen Balbach Udo Heinemann 《Acta Crystallographica. Section D, Structural Biology》2004,60(4):755-757
The cold‐shock response has been described for several bacterial species. It is characterized by distinct changes in intracellular protein patterns whereby a set of cold‐shock‐inducible proteins become abundant. The major cold‐shock proteins of Bacillus subtilis (Bs‐CspB) and Bacillus caldolyticus (Bc‐Csp) are small oligonucleotide/oligosaccharide‐binding (OB) fold proteins that have been described as binding single‐stranded nucleic acids. Bs‐CspB (Mr = 7365) and Bc‐Csp (Mr = 7333) were crystallized in the presence of the deoxyhexanucleotide (dT)6. Crystals of (dT)6 with Bs‐CspB grew in the orthorhombic space group C2221, with unit‐cell parameters a = 49.0, b = 53.2, c = 77.0 Å. Crystals with Bc‐Csp grew in the primitive orthorhombic space group P21212, with unit‐cell parameters a = 74.3, b = 64.9, c = 31.2 Å. These crystals diffract to maximal resolutions of 1.78 and 1.29 Å, respectively. The presence of protein and DNA in the crystals was demonstrated by Raman spectroscopy. 相似文献
10.
The interactions between 2‐mercaptoethanol, dimercaprol and CdSe quantum dots (QDs) in organic media have been investigated by spectral methods. The results showed that the fluorescence (FL) emission of CdSe QDs gradually decreased, with a slight red‐shift, after adding thiols to CdSe QDs solutions. With the increase of the concentrations of thiols, the resonance light scattering (RLS) signal of CdSe QDs had been strongly enhanced in the wavelength range 300–500 nm, which was confirmed by the formation of larger CdSe QDs particles. The effect of thiols on the FL emission of CdSe QDs could be described by a Stern–Volmer‐type equation with the concentration ranges 1.0 × 10–6–7.5 × 10–4 mol/L for 2‐mercaptoethanol and 1.0 × 10–7–2.5 × 10–5 mol/L for dimercaprol. The possible mechanism of the interaction was proposed according to the results of UV‐vis absorption and micro‐Raman spectroscopy. The results indicated that FL quenching was mainly attributable to the exchange of the QDs surface molecules. Copyright © 2008 John Wiley & Sons, Ltd. 相似文献
11.
Heydarkhan-Hagvall S Helenius G Johansson BR Li JY Mattsson E Risberg B 《Journal of cellular biochemistry》2003,89(6):1250-1259
Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors. 相似文献
12.
13.
A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development. Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers. The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively. The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1%. Separate cultures of these two fractions were compared with cocultures of both fractions. In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture. By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture. The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I. In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies. On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone. These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis. The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro. Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell-cell interactions during liver development. 相似文献
14.
Oleinikov V Sukhanova A Mochalov K Ustinova O Kudelina I Bronstein I Nabiev I 《Biopolymers》2002,67(6):369-375
We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex. 相似文献
15.
S. Bojanowski A. Fiedler A. Frank E. A. Stadlbauer G. Schilling 《Engineering in Life Science》2007,7(6):593-598
Anaerobic low temperature conversion (LTC) converts organic residues such as animal meal or meat and bone meal (MBM) to bio‐crude, a solid product, containing carbon and phosphorus, reaction water and non‐condensable gases. The yield of bio‐crude increases with the content of volatile solids. The efficiency of the conversion as well as the calorific value of the liquid fuel produced are favorably affected by the partial recycling of inorganic constituents, high amounts of volatile solids and a low percentage of heteroatoms present in the feeding material. Heating values are 32.3 MJ/kg for bio‐crude from animal meal and 19.5 MJ/kg for bio‐crude from MBM. Both bio‐crude and animal fat produced were effectively converted in a vertical reactor construction with a fixed bed of aluminosilicates of the zeolite family or acidic clays, respectively. Products are bio‐fuels of varying chemical qualities. Depending on the reaction temperature and the catalyst type, aliphatic hydrocarbons (T = 400 °C, ～97 %) or alkylbenzenes (T = 550 °C) are the main products. The calorific values of these bio‐fuels are in a range from 40.1 to 41.9 MJ/kg and the kinematic viscosities are between 0.9 and 2.29 mm2/s. The solid products of LTC from different biomass (sludge, animal meal, MBM) contain a significant amount of phosphorus. In the case of the solid product from MBM it was as high as 242 mg P2O5/g. Solubility in citric acid showed that in the case of MBM, 98.8 % of total phosphorus is potentially available to plants. Pot experiments demonstrated a similar plant growth as with other organic fertilizers. 相似文献
16.
The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester. 相似文献
17.
Eliana Cordero Jan Rüger Dominik Marti Abdullah S. Mondol Thomas Hasselager Karin Mogensen Gregers G. Hermann Jürgen Popp Iwan W. Schie 《Journal of biophotonics》2020,13(2)
Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor. 相似文献
18.
Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells. 相似文献
19.
We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm?1, the Trp bands at 870 and 877 cm?1, and the bands at 2940 (CH stretching) and 3420 cm?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015. 相似文献
20.
Xuejing Li Erzao Zeng Huixia Di Qiang Li Jingwei Ji Jie Yang Dingbin Liu 《Advanced Biosystems》2019,3(7)
Profiling cellular biomarkers without the interference of endogenous signals could facilitate the investigation of complex intracellular biological events and provide new possibilities for precision disease diagnosis. Herein, a surface‐enhanced Raman scattering (SERS) probe with a high signal‐to‐background ratio (SBR) for cellular biomarker imaging is constructed. The probes are prepared by incorporating Prussian blue (PB) with porous gold nanoparticles (p‐Au NPs). Due to their rich built‐in Raman hotspots, the p‐Au NPs are excellent SERS substrates that can significantly amplify the signals of the incorporated PB. In parallel, PB shows a single peak in the cellular silent region, where the signals from the probes and endogenous molecules can be completely resolved without the need of complex spectral unmixing. As a consequence, the combination of probe signal enhancement and background elimination endows the SERS probes with an extremely high SBR. To evaluate their performance in biomarker imaging, the high‐SBR SERS probes are utilized to profile folic acids at a single‐cell level. This background‐free, high‐precision imaging technique is conducive to early diagnosis and therapeutic response of cancer that is of great importance in clinical settings. 相似文献