Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway.     

Exercise as a model to study redox homeostasis in blood: the effect of protocol and sampling point

Twenty males ran either on a level treadmill (nonmuscle-damaging condition) or on a downhill treadmill (muscle-damaging condition). Blood and urine samples were collected before and after exercise (immediately after, 1h, 4h, 24h, 48h, and 96h). The following assays were performed: F(2)-isoprostanes in urine, protein carbonyls in plasma, glutathione, superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes. The main finding was that monophasic redox responses were detected after nonmuscle-damaging exercise compared to the biphasic responses detected after muscle-damaging exercise. Based on these findings, muscle-damaging exercise may be a more appropriate experimental model to induce physiological oxidative stress.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.The initiation of DNA replication for most double-stranded DNA (dsDNA)⁶ viral genomes begins with the recognition of the origin by specific origin-binding proteins. The herpes simplex virus type 1 (HSV-1) genome encodes seven proteins required for origin-dependent DNA replication. These are the DNA polymerase (UL30) and its accessory protein (UL42), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), the single-stranded DNA-binding protein (ICP8 or UL29), and the origin-binding protein (UL9) (reviewed in Ref. 1). HSV-1 contains three functional origins, oriL and two copies of oriS. OriS, which is about 80 bp in length, consists of three UL9 recognition sites, in Boxes I, II, and III, which are arranged in two overlapping palindromes (2). Box I and Box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in single-stranded DNA, which may be important in the origin rearrangement (3) during initiation of replication. Box I and II are separated by an AT-rich spacer sequence, which varies in length and nucleotide composition between the different members of the α-herpesvirus subfamily (2, 4–6).UL9 is a homodimer in solution, and EM studies, with UL9 bound to oriS, indicate the existence of a dimer or pair of dimers assembled on oriS (7). Several reports indicate that UL9 can physically interact not only with ICP8 (8) but also with other members of the HSV-1 replication complex, including UL8 (9) and UL42 (10). Thus UL9 functions as a docking protein to recruit these essential replication proteins to the viral origins. ICP8 stimulates the helicase activity of UL9 (11, 12) and binds to its C-terminal 27-aa residues (13). In the presence of ICP8, UL9 will open dsDNA containing Box I, leading to a conformational change in the origin, thus facilitating unwinding (14–16). As stated above, the changes in DNA conformation in the complete oriS may be more complex (3). Recently, it has been suggested that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by UL9. oriS* contains a hairpin formed by complementary base pairing between Box I and Box III in oriS (17). UL9, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-bp double-stranded minimal oriS fragment to oriS* and form a UL9-oriS* complex. The formation of a UL9-oriS* complex requires ATP hydrolysis (18). Therefore, the UL9-oriS* complex may serve as an assembly site for the herpesvirus replisome. Macao et al. (3) proposed a model in which full-length UL9 would be required to adopt a different conformation when binding to oriS or oriS*. The implication is that UL9 partially unwinds and introduces a hairpin into the origin of replication and that the formation of oriS* is aided, in some way, by ICP8 and requires ATP hydrolysis. Macao et al. (3) suggest that the length of the single-stranded tail of the probe DNA determines the stoichiometry of the UL9-DNA complex. oriS may bind two molecules of UL9, whereas oriS* may only bind one because the hairpin formation prevents the second interaction.Photo-cross-linking studies have shown that, although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I, suggesting that the C terminus of UL9 undergoes a conformational change upon binding to Box I (19). The results reported here are consistent with this observation. To date there is no three-dimensional structural information available on the full-length UL9 or either of the functionally characterized (helicase and DNA binding) domains. The ability to adopt different conformations and a tendency to proteolytic degradation may be responsible for this. It has been shown that UL9 binds with very high specificity to the Box I through its DNA-binding domain, consisting of the C-terminal 317 aa (UL9ct) (20, 21). Although the importance of the binding between UL9ct and oriS for the viral life cycle is well established, the mechanism behind this interaction still remains unclear. Even though UL9ct exists as a monomer in solution, uncertainty remains as to whether one or two molecules bind to a single Box I recognition sequence. Some reports have suggested that one UL9ct molecule binds to a single copy of the sequence (22–24), whereas others have proposed that UL9ct forms a dimer when bound to DNA (25, 26). This apparent difference may well result from the different protein concentrations used in different assays/experiments, which in turn highlights the difficulty of translating in vitro equilibrium experiments into cellular nonequilibrium situations.A few years ago, the crystal structure of a 60-residue C-terminal deletion mutant of ICP8 (ICP8ΔC) was determined to 3 Å resolution (Protein Data Bank code 1URJ (27)). The structure of ICP8ΔC consists of a large N-terminal domain (aa 9–1038) and a smaller entirely helical C-terminal domain (aa 1049–1120) connected to the N-terminal domain by a disordered linker (aa 1038–1049) spanning around 18 Å in the crystal structure. ICP8 preferentially binds ssDNA over dsDNA in a nonsequence-specific and cooperative manner (28). ICP8 is a zinc metalloprotein containing one zinc atom per molecule, which is coordinated by three cysteines (Cys-499, Cys-502, and Cys-510) and a histidine (His-512) (27).In this study, we show that the 60-amino acid C-terminal deletion of ICP8 (ICP8ΔC) binds strongly to UL9ct. We present three low resolution structures in solution using small angle x-ray scattering as follows: that of the UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer dsDNA (dsDNA_15-mer) containing the Box I sequence. Using these data and a variety of biophysical techniques, we demonstrate that the stoichiometries of the UL9ct-dsDNA_15-mer and UL9ct-ICP8ΔC-dsDNA_15-mer complexes are 2:1 and 2:1:1, respectively, at the micromolar protein concentrations used in this study. Using EM we visualize the assembly of the ICP8ΔC-UL9ct complex on oriS and estimate the size of the complex.     

Increases in serum concentration of human heart-type fatty acid-binding protein following elective coronary intervention

Background: Heart-type fatty acid-binding protein (H-FABP) is considered a marker of myocardial necrosis but whether or not it is modified by myocardial ischemia is not clear. We sought to investigate if H-FABP serum levels increase following non-urgent coronary angioplasty.

Methods: We studied 31 patients undergoing coronary angioplasty. Peripheral venous samples were drawn immediately before angioplasty, 1?h after the first balloon inflation and 24?h after the procedure and assayed for H-FABP.

Results: Serum levels of H-FABP increased significantly at 1?h vs baseline from 2554?±?1268 to 3322?±?245?pg?ml^?1 (p?=?0.024). However, no differences were observed between 1?h and 24?h after angioplasty (3268?±?1861 vs 3322?±?2459?pg ml^?1, p?=?0.87). Moreover, no significant difference was observed when we compared 24?h after angioplasty with the baseline (3268?±?1861vs 2554?±?1268?pg ml^?1, p?=?0.112).

Conclusions: We conclude that H-FABP significantly increases after elective coronary angioplasty at 1?h compared with baseline values; whether or not this has any prognostic significance for future events, as it occurs with troponins, needs to be studied further.     

Syncytins expression in cultured trophoblast cells according to differentiation status

Background  

Data of syncytin 1 and 2 env gene expression in human placenta and participation in the syncytialisation phenomena has been reported. However, there are not many studies on simultaneous changes in expression of both syncytins in culture. We sought evidence on the relative expression of syncytins and syncytin 1 receptors in trophoblast cell culture treated with a differentiation inducing factor (forskolin).     

Study of changes in physicochemical and microbiological characteristics of shrimps (Melicertus kerathurus) stored under modified atmosphere packaging

Fresh minimally processed shrimps were stored under modified atmosphere packaging (60% CO₂:40% N₂ for MAP A and 92.9% N₂:5.1% CO₂:2% O₂ for MAP B) for 5 days at 3 °C. Total mesophiles, H₂S forming bacteria, Pseudomonas spp., Brochothrix thermosphacta, firmness, color and sensory parameters were investigated throughout the whole time of the experiment. During storage period samples stored under MAP B managed to retain firmness values close to the initial values. All microbial populations growth was suppressed by the presence of MAP A. Samples stored under MAP B managed to maintain their firmness values close to the initial ones while MAP A samples were significantly less firm (p < 0.05).     

Resveratrol-Induced Vascular Progenitor Differentiation towards Endothelial Lineage via MiR-21/Akt/β-Catenin Is Protective in Vessel Graft Models

Background and Purpose

Vessel graft failure is typically associated with arteriosclerosis, in which endothelial dysfunction/damage is a key event. Resveratrol has been shown to possess cardioprotective capacity and to reduce atherosclerosis. We aimed to study the influence of resveratrol on the behavior of resident stem cells that may contribute to graft arteriosclerosis.

Experimental Approach

Vascular resident progenitor cells and embryonic stem cells were treated with resveratrol under differentiating conditions and endothelial markers expression was evaluated. Expression of miR-21 and β-catenin was also tested and exogenously modified. Effects of resveratrol treatment in an ex vivo re-endothelialization model and on mice undergone vascular graft were evaluated.

Key Results

Resveratrol induced expression of endothelial markers such as CD31, VE-cadherin and eNOS in both progenitor and stem cells. We demonstrated that resveratrol significantly reduced miR-21 expression, which in turn reduced Akt phosphorylation. This signal cascade diminished the amount of nuclear β-catenin, inducing endothelial marker expression and increasing tube-like formation by progenitor cells. Both the inhibition of miR-21 and the knockdown of β-catenin were able to recapitulate the effect of resveratrol application. Ex vivo, progenitor cells treated with resveratrol produced better endothelialization of the decellularized vessel. Finally, in a mouse model of vessel graft, a resveratrol-enhanced diet was able to reduce lesion formation.

Conclusions and Implications

We provide the first evidence that oral administration of resveratrol can reduce neointimal formation in a model of vascular graft and elucidated the underpinning miR-21/Akt/β-catenin dependent mechanism. These findings may support the beneficial effect of resveratrol supplementation for graft failure prevention.     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.