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Biophysical Studies (biophysical + studies)
Selected AbstractsBiophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein BFEBS JOURNAL, Issue 9 2000Deborah K. Wilkins The peptide CspB-1, which represents residues 1,22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water [M. Großet al. (1999) Protein Science8, 1350,1357] We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed. Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils. A CD spectrum indicative of ,,structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases. The characteristic wavelength shift of the amyloid-binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform-infrared spectrum indicative of ,,structure. Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well-defined amyloid fibrils are established on a timescale of hours. NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process. A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease-related systems. These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins. [source] Mechanisms of morphogen movementDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2005Maura Strigini Abstract Morphogens are defined as signaling molecules that are produced locally, yet act directly at a distance to pattern the surrounding field of cells in a concentration-dependent manner. In recent years many laboratories have devoted their attention to how morphogens actually reach distant cells. Several models have been proposed, including diffusion in the extracellular space and planar transcytosis. A combination of genetic, developmental, and cell-biological approaches have been taken to tackle this issue. I will present the models and discuss the types of experiments that have been designed to test them. It stands out that most of the work has been carried out in Drosophila. Morphogens contribute to patterning of the vertebrate nervous system, and the same signaling molecules have recently been shown to play important, possibly instructive, roles in axon guidance. Little, if anything, is known about the movement of morphogens in the context of nervous system development. The long-standing tradition of biophysical studies on diffusion in the brain extracellular space, along with the sophisticated in vitro culture systems developed in neurobiology laboratories, may provide new tools and ideas to test these models in a new context. © 2005 Wiley Periodicals, Inc. J Neurobiol 64: 324,333, 2005 [source] Circuitry of nuclear factor ,B signalingIMMUNOLOGICAL REVIEWS, Issue 1 2006Alexander Hoffmann Summary:, Over the past few years, the transcription factor nuclear factor (NF)-,B and the proteins that regulate it have emerged as a signaling system of pre-eminent importance in human physiology and in an increasing number of pathologies. While NF-,B is present in all differentiated cell types, its discovery and early characterization were rooted in understanding B-cell biology. Significant research efforts over two decades have yielded a large body of literature devoted to understanding NF-,B's functioning in the immune system. NF-,B has been found to play roles in many different compartments of the immune system during differentiation of immune cells and development of lymphoid organs and during immune activation. NF-,B is the nuclear effector of signaling pathways emanating from many receptors, including those of the inflammatory tumor necrosis factor and Toll-like receptor superfamilies. With this review, we hope to provide historical context and summarize the diverse physiological functions of NF-,B in the immune system before focusing on recent advances in elucidating the molecular mechanisms that mediate cell type-specific and stimulus-specific functions of this pleiotropic signaling system. Understanding the genetic regulatory circuitry of NF-,B functionalities involves system-wide measurements, biophysical studies, and computational modeling. [source] Influence of environmental stress on skin tone, color and melanogenesis in Japanese skinINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2005K. Kikuchi Introduction It is needless to say that one of the most potent environmental stress for melanogenesis of the human skin is the effect of ultraviolet (UV) light from the sunlight. Characteristic skin aging as a result of this UV light is recognized as photoaging. Clinical features in photoaging are wrinkles, skin laxity, coarseness, leathery, yellowing, lentigenes, mottled pigmentation, telangiectasia, sebaceous hypertrophy and purpura. There is an apparent difference in clinical features in photoaging among different races, i.e. between Caucasians, African American and Asians that include Japanese. Not only photo skin type but also environmental factors, such as climate, latitude, altitude and their habit of sunbathing, smoking and skin care influence the characteristic development of their photoaging. Racial difference in photoaging Caucasians tend to develop skin laxity and fine wrinkles more than Asians [1]. Asians tend to produce coarser wrinkles than the Caucasians although their development is rather late in life. There is also a difference in the skin color. Pigmentation is an earliest and prominent skin changes in Asians [1] and it increases with age [2]. In contrast, pigmentation is not apparent in the Caucasians although redness probably because of an increase in cutaneous vascularization becomes prominent in middle aged Caucasians [2]. Chung reported that seborrheic keratosis is a major pigmentary lesion in men, whereas hyperpigmented macules are prominent features in women in Koreans [3]. Melanogenesis pigmentation disorders in Japanese Ephelides (freckles) are commonly found in those with photo skin type I who have fair skin and red eyes and blond hair. They are also found in the Japanese. Clinical feature reveals that multiple small pigmentary macules on sun-exposed areas mainly on the mid-portion of the face. These lesions seem to be familial, becoming apparent even in early childhood after sun exposure. Melasma is an acquired pigmentary disorder commonly found in middle aged Japanese women characterized by irregular brown macules and patches on the sun-exposed areas on the face typically as bilaterally present macules on the cheeks. An increase in sex hormones as a result of pregnancy and intake of contraceptive pills is one of the etiological factors to develop melasma. Sun exposure also worsens it. Nevus of Ota is also a common pigmentary disorder found in the Japanese. It is usually unilateral, blue-brown to slate-gray pigmentary macules on the eyelid and cheek that appear in early childhood or in puberty. Acquired dermal melanocytosis is also a pigmentary disorder, in which dermal melanocytes are found as shown in nevus of Ota, characterized by bilateral brown to blue-gray macules on the forehead, temple, eyelid and malar areas in middle aged Japanese women. This tends to be misdiagnosed as melasma. Solar lentigo is an acquired pigmented macule induced by sun exposure. Solar lentigines are usually multiple, circumscribed brown macules. There are two types of solar lentigo. One is a small macular type, characterized by multiple, small brown macules whose diameter is less than 5 mm, being similar to ephelides (freckles). The other type is a large macular type, characterized by a few round to oval, brown macules whose diameter is beyond 1 cm. Some of their surface are hyperkeratotic and become elevated to produce seborrheic keratosis. Again, the early sign of photoaging in Japanese is pigmentated spots and these pigmentation disorders increase with age. Among the pigmentary changes, nevus of Ota, acquired dermal melanocytosis, melasma and large macular type of solar lentigo are characteristic skin changes found in the Japanese in addition to ephelides and small macular type of solar lentigo. Seasonal changes of the various functional properties of the skin including skin color assessed by non-invasive bioengineering techniques [4]. When we consider skin tone, color and melanogenesis, UV light from the sunlight is the most potent environmental stress, although we cannot forget also the important influence of environmental relative humidity affects our skin functions as well as its appearance. We investigated seasonal influences on the various properties of the skin in 39 healthy Japanese females consisting of different age groups. Their skin is thought to be affected by the UV light in summer, and by the exposure to the dry and cold air in winter. Materials and methods Biophysical, non-invasive measurements, including transepidermal water loss (TEWL) as a parameter for the barrier function of the stratum corneum (SC), high frequency conductance as a parameter for the hydration state of the SC, skin color and casual surface lipid levels, were conducted during late summer and winter months. Skin color was determined with a chromameter according to the L*a*b* CIE 1976 system, where L* is an attribute on the luminance scale, a* that on the red versus green scale and b* that on the yellow versus blue scale. All the measurements were conducted in an environment controlled-chamber (21 ± 1 °C room temperature, and 50 ± 3% relative humidity). Results The barrier function of the SC was found to be significantly impaired in winter on the cheek as compared with that measured in summer, whereas no such seasonal change was apparent both in the hydration state of the SC and sebum levels on the cheek. In the assessment of the skin color on the cheek, a significant increase in a* (redness) and a decrease in b* (yellowness) were observed in winter. In contrast, on the flexor forearm, the values of L* (luminescence) increased in winter, but no seasonal change was noted in the values of a* and b*. In this study, skin changes with aging were also found by the non-invasive bioengineering methods. The value of TEWL on the cheek tended to increase with age, whereas no significant change was observed in the value of TEWL on the forearm. In the assessment of skin color, b* value on the cheek significantly increased with age whereas a* and L* values on the cheek did not show any significant change with age. Summary of this study We think that such an increase in yellowness with aging of the cheek skin is a phenomenon unique to the Japanese (Asians) since an increase in b* value was not observed in Caucasians [2]. The facial skin that is always exposed shows barrier impairment in a dry and cold winter environment and demonstrates increased yellowness in skin color because of a prolonged exposure to the UV light from the sun in the summer season. The non-invasive bioengineering methods are useful to demonstrate even invisible seasonal changes occurring in the same individuals and changes with age occurring in the skin. References 1.,Goh, S.H. The treatment of visible signs of senescence: the Asian experience. Br. J. Dermatol.122, 105,109 (1990). 2.,LeFur, I., Numagami, K., Guinot, C. et al. Age-related reference values of skin color in Caucasian and Japanese healthy women according to skin site. Pigment Cell Res. 7, 67 (1999). 3.,Chung, J.H., Lee, S.H., Youn, C.S. et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch. Dermatol. 137, 1043,1051 (2001). 4.,Kikuchi, K., Kobayashi, H., Le Fur, I. et al. Winter season affects more severely the facial skin than the forearm skin: comparative biophysical studies conducted in the same Japanese females in later summer and winter. Exog. Dermatol. 1, 32,38 (2002). [source] Functional implications for Kir4.1 channels in glial biology: from K+ buffering to cell differentiationJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Michelle L. Olsen Abstract Astrocytes and oligodendrocytes are characterized by a very negative resting potential and a high resting permeability for K+ ions. Early pharmacological and biophysical studies suggested that the resting potential is established by the activity of inwardly rectifying, Ba2+ sensitive, weakly rectifying Kir channels. Molecular cloning has identified 16 Kir channels genes of which several mRNA transcripts and protein products have been identified in glial cells. However, genetic deletion and siRNA knock-down studies suggest that the resting conductance of astrocytes and oligodendrocytes is largely due to Kir4.1. Loss of Kir4.1 causes membrane depolarization, and a break-down of K+ and glutamate homeostasis which results in seizures and wide-spread white matter pathology. Kir channels have also been shown to act as critical regulators of cell division whereby Kir function is correlated with an exit from the cell cycle. Conversely, loss of functional Kir channels is associated with re-entry of cells into the cell cycle and gliosis. A loss of functional Kir channels has been shown in a number of neurological diseases including temporal lobe epilepsy, amyotrophic lateral sclerosis, retinal degeneration and malignant gliomas. In the latter, expression of Kir4.1 is sufficient to arrest the aberrant growth of these glial derived tumor cells. Kir4.1 therefore represents a potential therapeutic target in a wide variety of neurological conditions. [source] Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosisPROTEIN SCIENCE, Issue 10 2010Chi Wang Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source] Mutations of key hydrophobic surface residues of 11,-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression systemPROTEIN SCIENCE, Issue 7 2009Alexander J. Lawson Abstract 11,-Hydroxysteroid dehydrogenase type 1 (11,-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11,-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered Km values for steroids and an unaltered or increased kcat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild-type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme. [source] Atomic-resolution crystal structure of thioredoxin from the acidophilic bacterium Acetobacter acetiPROTEIN SCIENCE, Issue 1 2007Courtney M. Starks Abstract The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 Å resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization. [source] Facile chemical synthesis and equilibrium unfolding properties of CopGPROTEIN SCIENCE, Issue 7 2004Thomas E. Wales Abstract The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, ,Gf and m -values of ,13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG. [source] Liposome-bound APO2L/TRAIL is an effective treatment in a rabbit model of rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 8 2010Luis Martinez-Lostao Objective We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA). Methods We used a novel Ni2+ -(N -5-amino-1-carboxypentyl)-iminodiacetic acid),containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed. Results Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic. Conclusion Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA. [source] ,-Crustacyanin, the blue,purple carotenoprotein of lobster carapace: consideration of the bathochromic shift of the protein-bound astaxanthinACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2003P. F. Zagalsky The crystal structure of a ,-crustacyanin allows an analysis of the various proposals for the mechanism of the bathochromic shift from orange to purple,blue of astaxanthin in this lobster carotenoprotein. Structural and previous chemical and biophysical studies suggest that extension of conjugation by coplanarization of the ,-ionone rings with the polyene chain and polarization resulting from hydrogen bonding at the C(4) and C(4,) keto groups may be mainly responsible for the bathochromic shift. Additional contributions may arise from medium effects and possibly from bowing of the polyene chain on binding. Previous biophysical data revealing a somewhat symmetrical polarization of astaxanthin in crustacyanin are thereby also accounted for. A puzzling feature remains unexplained: the bathochromic shifts, larger than that of astaxanthin, shown by some cyclopentenedione carotenoids in reconstituted carotenoproteins. This mini review enlarges on the original analysis and conclusions of Cianci et al. [(2002), Proc. Natl Acad. Sci. USA, 99, 9795,9800]. [source] Labeling of proteins with fluorescent probes: Photophysical characterization of dansylated bovine serum albumin,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2003Valeria Levi Abstract Fluorescence spectroscopy is a widely used technique in biophysical studies. One of the strategies frequently used consists of labeling biomolecules with fluorescent probes, which have distinctive photophysical properties. This methodology allows the study of a wide variety of structural features of the biomolecule. We describe a simple laboratory activity for undergraduate Biophysical Chemistry courses. The experimental work includes two activities: labeling BSA with dansyl chloride and analyzing the resulting absorption and fluorescence spectra. The discussion of these activities helps students to understand the basis of fluorescence spectroscopy with emphasis in the application to biological systems. [source] X-ray structure and biophysical properties of rabbit fibroblast growth factor 1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Jihun Lee The rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel pro-angiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the biophysical properties of rabbit FGF-1 are described. The X-ray structure shows that the amino-acid differences between human and rabbit FGF-1 are solvent-exposed and therefore potentially immunogenic, while the biophysical studies identify differences in thermostability and receptor-binding affinity that distinguish rabbit FGF-1 from human FGF-1. [source] Structure of the ligand-binding domain of the EphB2 receptor at 2,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Yehuda Goldgur Eph tyrosine kinase receptors, the largest group of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell,cell communication regulating cell attachment, shape and mobility. Recently, several Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Structural and biophysical studies have established detailed information on the binding and recognition of Eph receptors and ephrins. The initial high-affinity binding of Eph receptors to ephrin occurs through the penetration of an extended G,H loop of the ligand into a hydrophobic channel on the surface of the receptor. Consequently, the G,H loop-binding channel of Eph receptors is the main target in the search for Eph antagonists that could be used in the development of anticancer drugs and several peptides have been shown to specifically bind Eph receptors and compete with the cognate ephrin ligands. However, the molecular details of the conformational changes upon Eph/ephrin binding have remained speculative, since two of the loops were unstructured in the original model of the free EphB2 structure and their conformational changes upon ligand binding could consequently not be analyzed in detail. In this study, the X-ray structure of unbound EphB2 is reported at a considerably higher 2,Å resolution, the conformational changes that the important receptor loops undergo upon ligand binding are described and the consequences that these findings have for the development of Eph antagonists are discussed. [source] | ||||||||||||||||