Determining denaturation midpoints in multiprobe equilibrium protein folding experiments

Multiprobe equilibrium unfolding experiments in the downhill regime (i.e., maximal barrier < 3 RT) can resolve the folding process with atomic resolution [ Munoz ( 2002) Int. J. Quantum Chem. 90, 1522 -1528] . Such information is extracted from hundreds of heterogeneous atomic equilibrium unfolding curves, which are characterized according to their denaturation midpoint (e.g., T m for thermal denaturation). Using statistical methods, we analyze T m accuracy when determined from the extremum of the derivative of the unfolding curve and from two-state fits under different sets of simulated experimental conditions. We develop simple procedures to discriminate between real unfolding heterogeneity at the atomic level and experimental uncertainty in the single T m of conventional two-state folding. We apply these procedures to the recently published multiprobe NMR experiments of BBL [ Sadqi et al. ( 2006) Nature 442, 317 -321 ] and conclude that for the 122 single transition atomic unfolding curves reported for this protein the mean T m accuracy is better than 1.8 K for both methods, compared to the 60 K spread in T m determined experimentally. Importantly, we also find that when the pre- or posttransition baseline is incomplete, the two-state fits systematically drift the estimated T m value toward the center of the experimental range. Therefore, the reported 60 K T m spread in BBL is in fact a lower limit. The derivative method is significantly less sensitive to this problem and thus is a better choice for multiprobe experiments with a broad T m distribution. The results we obtain in this work lay the foundations for the quantitative analysis of future multiprobe unfolding experiments in fast-folding proteins.     

Impact of Postovulatory Food Deprivation on the Ova Transport, Hormonal Profiles and Metabolic Changes in Sows

The effect of food deprivation on ova transport, hormonal profiles and metabolic changes was studied in 20 crossbred multiparous sows during their second oestrus after weaning. To determine the time of ovulation, transrectal ultrasonographic examination was performed. The sows were divided into 2 groups, one control group (C-group), which was fed according to Swedish standards, and one experimental group (E-group). The E-group sows were deprived of food from the first morning meal after ovulation until slaughter. Blood samples were collected every second hour from about 12 h before expected ovulation in the second oestrus after weaning until slaughter and were analysed for progesterone, prostaglandin F_2α-metabolite, insulin, glucose, free fatty acids and triglycerides. All sows were slaughtered approximately 48 h after ovulation and the genital tract was recovered. The isthmic part of the oviduct was divided into 3 equally long segments and flushed separately with phosphate buffered saline (PBS). Uterine horns were also flushed with PBS. A significantly greater number of ova were found in the first and second part of the isthmus in the E-group (p = 0.05) while in the C-group most of the ova were found in the third part of the isthmus or the uterus (p = 0.01). The level of prostaglandin F_2α-metabolite was significantly higher in the E-group compared with the C-group. The concentration of progesterone increased in both groups after ovulation but there were no significant differences between the groups. The other blood parameters showed that the food-deprived sows were in a catabolic state. The 48 h period of fasting results, directly or indirectly in an delayed ova transport, which may be due to a delayed relaxation in the smooth circular muscle layer of the isthmus.     

Production of Melanocyte‐Specific Antibodies to Human Melanosomal Proteins: Expression Patterns in Normal Human Skin and in Cutaneous Pigmented Lesions

Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide‐specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross‐reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte‐specific markers, tyrosinase, tyrosinase‐related protein 1 (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.     

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

Background  

Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIα, and to the nucleolus.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitro and in vivo as well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Causes,historical development,effects and future challenges of a common environmental problem: eutrophication

The impact of the presence of us humans as well as our activities to nature has led to over-exploitation of natural resources and to dramatic changes in land-use including the use of artificial fertilizers contributes to the deterioration of the natural environment. The population density, industrial processes and the use of fertilizers are the main causes for the eutrophication of river systems, estuaries and seas. There are several ways to determine the actual nutrient levels back to the 1950s, back to 1900 or even back to 1800. Available data indicate that the natural background concentrations of nutrients (pristine conditions or the period prior to the widespread use of artificial fertilizers and detergents) were dramatically lower than today. Available time series on chemical and biological data collected from different parts of the world show the (sometimes tremendous) increase in nutrient levels and the related productivity. The same time series, however, also show the decreases in values when measures were taken to reduce the nutrient emissions. Investigations of different systems all over the world show that nearly every system responses differently to eutrophication. Especially physical boundary conditions play an important role in the manifestation of the ultimate effect of local eutrophication. Apart from the physical boundary conditions also the transformation and retention of nutrients in estuarine and coastal systems contribute to system specific responses. Depending on all these different conditions, site specific responses with even site specific problems may occur. The challenge of this millennium is to really reach a balance between nature and mankind including its population size. A beneficial step in this discussion is assessing criteria to reduce eutrophication back to a level acceptable to both humans and nature.     

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.     

Association of Bcr-Abl with the proto-oncogene Vav is implicated in activation of the Rac-1 pathway.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1.