Crystal structure of venus,a yellow fluorescent protein with improved maturation and reduced environmental sensitivity

Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 A resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37 degrees C by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the Calpha-Cbeta bond of Tyr(66) during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site.     

Cardiomyocyte volume-weighted nuclear volume and spironolactone therapy in spontaneously hypertensive rats

OBJECTIVE: To evaluate the blood pressure (BP) and cardiomyocyte nuclei hypertrophy of spontaneously hypertensive rats (SHR) treated with spironolactone and with spironolactone and an angiotensin-converting enzyme inhibitor and calcium channel blocker. STUDY DESIGN: Six groups with 5 SHR each were treated for 13 weeks: control group, spironolactone groups (5, 10 and 30 mg/kg/d, or low, moderate and high doses, respectively), and spironolactone and enlapril or/and verapamil. Blood pressure (BP) and cardiomyocyte volume-weighted nuclear volume (VWNV) were studied. RESULTS: The usual evolution of BP in young adult SHR was greatly altered by spironolactone treatment, with either attenuation or reversion of the BP tendency to increase. The treatments efficiently reduced the cardiomyocyte VWNV > 45%, confirming efficient action of both spironolactone monotherapy at moderate and high dose and spironolactone combined with enalapril or verapamil. However, the high dose of spironolactone, and spironolactone plus enalapril or verapamil showed no significant differences in cardiomyocyte VWNV. Correlation between VWNV and BP (13th week) suggested that BP degree influences cardiomyocyte VWNV. CONCLUSION: The effect of spironolactone monotherapy seems to be dose dependent, and the combination with enalapril or verapamil improves spironolactone efficiency in BP reduction but not in prevention/attenuation of cardiac myocyte nuclear hypertrophy.     

Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments

Mismatch repair (MMR) genes, such as Msh2, are classified as "mutator" genes, responsible for the microsatellite instability identified in many tumors. In the current study, the mutation frequency and mutational spectrum in thymic lymphoma arising in Msh2 deficient mice are investigated. Thymic lymphoma developed in Msh2-/- background displayed an eight to nine-fold increase in mutation frequency compared to the normal thymi in Msh2 deficient animals. Sequencing demonstrated significantly different mutational spectra between normal thymus tissue and thymic lymphomas in Msh2-/- mice (P=0.02). The tumor mutational spectrum is characterized by an increase in base substitutions occurring at A:T sites, and multiple mutations, as well as a minor increase in -1 frameshifts. We analyzed mutations in different parts of the tumors, and different regional hotspots could be identified. Several hotspot mutations that are a rare event in normal tissues were identified in the tumor tissues. We conclude that thymic lymphomas arising in Msh2 deficient genetic background are hypermutable and the altered mutational spectrum might be an indication of infidelity of DNA replication during tumorigenesis.     

Gastroprotective effect of adrenomedullin administered subcutaneously in the rat

Subcutaneous injections of adrenomedullin prevented reserpine-induced gastric mucosal damage in a dose-dependent manner (1-1000 ng/kg), but did not interfere with the lesions produced by ethanol administration. In pylorus-ligated rats adrenomedullin significantly reduced gastric volume, total and free acid output as well as ulcer formation. The gastroprotective activity of adrenomedullin was not present in rats pretreated with cysteamine. These results suggest that adrenomedullin exerts its antiulcer effect, when it is administered subcutaneously (s.c.), probably by a mechanism which involves somatostatin related transmission.     

Different telomere damage signaling pathways in human and mouse cells

Programmed telomere shortening in human somatic cells is thought to act as a tumor suppressor pathway, limiting the replicative potential of developing tumor cells. Critically short human telomeres induce senescence either by activating p53 or by inducing the p16/RB pathway, and suppression of both pathways is required to suppress senescence of aged human cells. Here we report that removal of TRF2 from human telomeres and the ensuing de-protection of chromosome ends induced immediate premature senescence. Although the telomeric tracts remained intact, the TRF2(DeltaBDeltaM)-induced premature senescence was indistinguishable from replicative senescence and could be mediated by either the p53 or the p16/RB pathway. Telomere de-protection also induced a growth arrest and senescent morphology in mouse cells. However, in this setting the loss of p53 function was sufficient to completely abrogate the arrest, indicating that the p16/RB response to telomere dysfunction is not active in mouse cells. These findings reveal a fundamental difference in telomere damage signaling in human and mouse cells that bears on the use of mouse models for the telomere tumor suppressor pathway.     

The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.     

The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.     

Circulating VEGF and its soluble receptors sVEGFR-1 and sVEGFR-2 in patients with acute leukemia

Angiogenesis plays an important role in the pathogenesis of acute leukemia, and vascular endothelial growth factor (VEGF) is a crucial, positive regulator of this process. The biological activity of VEGF is mediated by two different receptor tyrosine kinases: VEGFR-2 and VEGFR-1. The soluble form of VEGFR-1 is likely to be a negative regulator of VEGF availability, but the physiological role of sVEGFR-2 is still unclear. The plasma levels of sVEGFR-1 and sVEGFR-2 in patients with acute leukemia have not been investigated. We measured the plasma concentrations of VEGF and its two soluble receptors in 39 AML and 15 ALL patients as well as in the control group, using the ELISA assay. We also correlated the plasma levels of these proteins with disease status and known prognostic factors. The sVEGFR-1 level was significantly higher in patients with AML and ALL than in the healthy subjects (p < 0.002 and p < 0.03 respectively). The sVEGFR-2 level was significantly higher in AML patients compared with the control group (p < 0.03). The VEGF levels in AML and ALL patients and in healthy subjects did not differ significantly. The sVEGFR-1 level was higher in AML patients with > 50% of blasts in the bone marrow (BM), WBC > 20 G/L and elevated LDH level, than in the group with BM blasts < 50% (p < 0.01), WBC < 20 G/L (p < 0.02) and a normal LDH level (p < 0.05). Positive correlations between sVEGFR-1 level and WBC (p < 0.02),% of BM blasts (p < 0.05), the absolute blast count in peripheral blood (ABC) (p < 0.009) and LDH (p < 0.000001) were found. The sVEGFR-1/VEGF ratio (R1) was calculated, and a positive correlation between R1 and ABC in AML (p < 0.03) was determined. A higher (above median) sVEGFR-1/VEGF ratio correlated with a lower CR rate and a shorter survival (p < 0.03 and p = 0.0007 respectively). In conclusion, the plasma concentration of sVEGFR-1 is higher in leukemia patients than in healthy subjects and correlates with tumour burden and poor prognosis. The sVEGFR-1/VEGF ratio may be of greater prognostic value than VEGF alone. Further investigation is recommended to better determine their function.     

Genetic basis of autosomal dominant nocturnal frontal lobe epilepsy

In this review the current literature regarding autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is presented and discussed. This disease is caused by mutations of genes coding for subunits of neuronal acetylcholine receptor comprising the sodium/potassium ion channel. To date, three types of mutations of the gene encoding alpha4 subunit of acetylcholine receptor were described in multi-generation families in Australia, Spain, Norway and Japan. Two other types of mutations of the beta2 subunit were also reported in two families, one from Italy and the other from Scotland. Mutations were caused by substitutions of a single nucleotide or by several-nucleotide insertions and result in a decrease or an increase in the activity of the receptor, or its changes in the affinity to the ligand. Recent advances in molecular genetics have provided the means for a better understanding of human epileptogenesis at a molecular level, which facilitates clinical diagnosis and provides a more rational basis of therapy of this form of epilepsy.