Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (βI-C1 and βII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid β-spectrin isoforms. We have identified interacting scFvs, with clones "G5" and "A2" binding only to βI-C1, and clone "F11" binding only to βII-C1. The K(d) values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 μM. A more quantitative K(d) value from isothermal titration calorimetry experiments with the recombinant G5 and βI-C1 was 0.15 μM. The α-spectrin fragments (model proteins), αI-N1 and αII-N1, competed with the βI-C1, or βII-C1, binding scFvs, with inhibitory concentration (IC(50) ) values of ～50 μM for αI-N1, and ～0.5 μM for αII-N1. Our predicted structures of βI-C1 and βII-C1 suggest that the Helix B' of the C-terminal partial domain of βI differs from that of βII. Consequently, an unstructured region downstream of Helix B' in βI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in βII was helical, and βII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different β-spectrin isoforms to regulate α- and β-spectrin association to form functional spectrin tetramers, and may sort β-spectrin isoforms to their specific cellular localizations.     

Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants

Effects of water stress on plants have been well-documented. However, the combined responses to drought and rewatering and their underlying mechanisms are relatively unknown. The present study attempts to describe spatiotemporal alterations in the physiology and cellular status of Medicago truncatula tissues that result from and subsequently follow a period of moderate water deficit. Physiological processes and cellular damage levels were monitored in roots and leaves by determining lipid peroxidation levels, as well as nitric oxide and hydrogen peroxide content, further supported by stomatal conductance and chlorophyll fluorescence measurements in leaves. During water stress, cells in both organs displayed increased damage levels and reactive oxygen and nitrogen species content, while leaves showed reduced stomatal conductance. Furthermore, both tissues demonstrated increased proline content. Upon rewatering, plants recovered displaying readings similar to pre-stress control conditions. Furthermore, molecular analysis of antioxidant gene expression by quantitative real-time RT-PCR revealed differential spatiotemporal regulation in a number of genes examined (including catalase, cytosolic ascorbate peroxidase, copper/zinc and iron superoxide dismutase and alternative oxidase). Overall, M. truncatula plants demonstrated increased sensitivity to drought-induced oxidative damage; however, this was reversed following rewatering indicating a great elasticity in the plant''s capacity to cope with free oxygen and nitrogen radicals.Key words: drought stress, antioxidants, cellular damage, medicago, proline, hydrogen peroxide, nitric oxide, reactive oxygen species, reactive nitrogen species     

Barrier passage and protein dynamics in enzymatically catalyzed reactions.

This review describes studies of particular enzymatically catalyzed reactions to investigate the possibility that catalysis is mediated by protein dynamics. That is, evolution has crafted the protein backbone of the enzyme to direct vibrations in such a fashion to speed reaction. The review presents the theoretical approach we have used to investigate this problem, but it is designed for the nonspecialist. The results show that in alcohol dehydrogenase, dynamic protein motion is in fact strongly coupled to chemical reaction in such a way as to promote catalysis. This result is in concert with both experimental data and interpretations for this and other enzyme systems studied in the laboratories of the two other investigators who have published reviews in this issue.     

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.     

Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell "stabilizer", is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 μM) can effectively inhibit secretion of histamine and PGD(2). Que and cromolyn also inhibit histamine, leukotrienes and PGD(2) from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.     

Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage

Introduction

The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.

Methods

Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.

Results

In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.

Conclusions

Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.     

TULIPs: tunable, light-controlled interacting protein tags for cell biology

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.     

Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4,992) of the DNA clones were found to be significantly different (P < 0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic- and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivo-produced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur.