A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.     

Potentiometric redox measurements in the environment: a novel perspective on method powers and limitations

Biogeochemistry - The South China Sea (SCS) is one of the largest marginal seas in the world, but the processes that control the silicon cycle are not well understood. Here, we analyse the factors...     

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.     

Defining the biological bases of individual differences in musicality

Advances in molecular technologies make it possible to pinpoint genomic factors associated with complex human traits. For cognition and behaviour, identification of underlying genes provides new entry points for deciphering the key neurobiological pathways. In the past decade, the search for genetic correlates of musicality has gained traction. Reports have documented familial clustering for different extremes of ability, including amusia and absolute pitch (AP), with twin studies demonstrating high heritability for some music-related skills, such as pitch perception. Certain chromosomal regions have been linked to AP and musical aptitude, while individual candidate genes have been investigated in relation to aptitude and creativity. Most recently, researchers in this field started performing genome-wide association scans. Thus far, studies have been hampered by relatively small sample sizes and limitations in defining components of musicality, including an emphasis on skills that can only be assessed in trained musicians. With opportunities to administer standardized aptitude tests online, systematic large-scale assessment of musical abilities is now feasible, an important step towards high-powered genome-wide screens. Here, we offer a synthesis of existing literatures and outline concrete suggestions for the development of comprehensive operational tools for the analysis of musical phenotypes.     

13C, 2H NMR Studies of Structural and Dynamical Modifications of Glucose-Exposed Porcine Aortic Elastin

Elastin, the principal component of the elastic fiber of the extracellular matrix, imparts to vertebrate tissues remarkable resilience and longevity. This work focuses on elucidating dynamical and structural modifications of porcine aortic elastin exposed to glucose by solid-state NMR spectroscopic and relaxation methodologies. Results from macroscopic stress-strain tests are also presented and indicate that glucose-treated elastin is mechanically stiffer than the same tissue without glucose treatment. These measurements show a large hysteresis in the stress-strain behavior of glucose-treated elastin—a well-known signature of viscoelasticity. Two-dimensional relaxation NMR methods were used to investigate the correlation time, distribution, and population of water in these samples. Differences are observed between the relative populations of water, whereas the measured correlation times of tumbling motion of water across the samples were similar. ¹³C magic-angle-spinning NMR methods were applied to investigate structural and dynamical modifications after glucose treatment. Although some overall structure is preserved, the process of glucose exposure results in more heterogeneous structures and slower mobility. The correlation times of tumbling motion of the ¹³C-¹H internuclear vectors in the glucose-treated sample are larger than in untreated samples, pointing to their more rigid structure. The ¹³C cross-polarization spectra reveal a notably increased α-helical character in the alanine motifs after glucose exposure. Results from molecular dynamics simulations are provided that add further insight into dynamical and structural changes of a short repeat, [VPGVG]₅, an alanine pentamer, desmosine, and isodesmosine sites with and without glucose. The simulations point to changes in the entropic and energetic contributions in the retractive forces of VPGVG and AAAAA motifs. The most notable change is the increase of the energetic contribution in the retractive force due to peptide-glucose interactions of the VPGVG motif, which may play an important role in the observed stiffening in glucose-treated elastin.     

Nuclear receptors in renal disease

Diabetes is the leading cause of end-stage renal disease in developed countries. In spite of excellent glucose and blood pressure control, including administration of angiotensin converting enzyme inhibitors and/or angiotensin II receptor blockers, diabetic nephropathy still develops and progresses. The development of additional protective therapeutic interventions is, therefore, a major priority. Nuclear hormone receptors regulate carbohydrate metabolism, lipid metabolism, the immune response, and inflammation. These receptors also modulate the development of fibrosis. As a result of their diverse biological effects, nuclear hormone receptors have become major pharmaceutical targets for the treatment of metabolic diseases. The increasing prevalence of diabetic nephropathy has led intense investigation into the role that nuclear hormone receptors may have in slowing or preventing the progression of renal disease. This role of nuclear hormone receptors would be associated with improvements in metabolism, the immune response, and inflammation. Several nuclear receptor activating ligands (agonists) have been shown to have a renal protective effect in the context of diabetic nephropathy. This review will discuss the evidence regarding the beneficial effects of the activation of several nuclear, especially the vitamin D receptor (VDR), farnesoid X receptor (FXR), and peroxisome-proliferator-associated receptors (PPARs) in preventing the progression of diabetic nephropathy and describe how the discovery and development of compounds that modulate the activity of nuclear hormone receptors may provide potential additional therapeutic approaches in the management of diabetic nephropathy. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.     

RTVP-1 expression is regulated by SRF downstream of protein kinase C and contributes to the effect of SRF on glioma cell migration

Gliomas are characterized by increased infiltration into the surrounding normal brain tissue. We recently reported that RTVP-1 is highly expressed in gliomas and plays a role in the migration of these cells, however the regulation of RTVP-1 expression in these cells is not yet described. In this study we examined the role of PKC in the regulation of RTVP-1 expression and found that PMA and overexpression of PKCα and PKCε increased the expression of RTVP-1, whereas PKCδ exerted an opposite effect. Using the MatInspector software, we identified a SRF binding site on the RTVP-1 promoter. Chromatin immunoprecipitation (ChIP) assay revealed that SRF binds to the RTVP-1 promoter in U87 cells, and that this binding was significantly increased in response to serum addition. Moreover, silencing of SRF blocked the induction of RTVP-1 expression in response to serum. We found that overexpression of PKCα and PKCε increased the activity of the RTVP-1 promoter and the binding of SRF to the promoter. In contrast, overexpression of PKCδ blocked the increase in RTVP-1 expression in response to serum and the inhibitory effect of PKCδ was abrogated in cells expressing a SRFT160A mutant. SRF regulated the migration of glioma cells and its effect was partially mediated by RTVP-1. We conclude that RTVP-1 is a PKC-regulated gene and that this regulation is at least partly mediated by SRF. Moreover, RTVP-1 plays a role in the effect of SRF on glioma cell migration.     

Rickettsia 'in' and 'out': two different localization patterns of a bacterial symbiont in the same insect species

Intracellular symbionts of arthropods have diverse influences on their hosts, and their functions generally appear to be associated with their localization within the host. The effect of localization pattern on the role of a particular symbiont cannot normally be tested since the localization pattern within hosts is generally invariant. However, in Israel, the secondary symbiont Rickettsia is unusual in that it presents two distinct localization patterns throughout development and adulthood in its whitefly host, Bemisia tabaci (B biotype). In the "scattered" pattern, Rickettsia is localized throughout the whitefly hemocoel, excluding the bacteriocytes, where the obligate symbiont Portiera aleyrodidarum and some other secondary symbionts are housed. In the "confined" pattern, Rickettsia is restricted to the bacteriocytes. We examined the effects of these patterns on Rickettsia densities, association with other symbionts (Portiera and Hamiltonella defensa inside the bacteriocytes) and on the potential for horizontal transmission to the parasitoid wasp, Eretmocerus mundus, while the wasp larvae are developing within the whitefly nymph. Sequences of four Rickettsia genes were found to be identical for both localization patterns, suggesting that they are closely related strains. However, real-time PCR analysis showed very different dynamics for the two localization types. On the first day post-adult emergence, Rickettsia densities were 21 times higher in the "confined" pattern vs. "scattered" pattern whiteflies. During adulthood, Rickettsia increased in density in the "scattered" pattern whiteflies until it reached the "confined" pattern Rickettsia density on day 21. No correlation between Rickettsia densities and Hamiltonella or Portiera densities were found for either localization pattern. Using FISH technique, we found Rickettsia in the gut of the parasitoid wasps only when they developed on whiteflies with the "scattered" pattern. The results suggest that the localization pattern of a symbiont may influence its dynamics within the host.     

Precocious puberty: growth and genetics

The precise neuroendocrine mechanisms underlying activation of hypothalamic-pituitary-gonadal (HPG) axis maturation are elusive. The wide age range of pubertal onset among normal individuals throughout the world may suggest that both genetic and environmental factors modulate the timing of puberty. Early activation of the HPG axis, termed central precocious puberty (CPP), causes psychosocial difficulties and may lead to compromised final height, especially if medical intervention is delayed. Although CPP is considered to be idiopathic in the majority of patients, we have recently reported a 27.5% prevalence of familial cases among 147 patients with idiopathic CPP. Segregation analysis of this cohort suggested an autosomal dominant transmission with incomplete sex-dependent penetrance. Allelic variants of candidate genes that regulate the timing of puberty may cause familial CPP. Detection of these genes will provide a tool for identification of children at risk of developing CPP, enabling early intervention with the aim of preventing its distressing outcomes.     

Human immunodeficiency virus (HIV)-specific gamma interferon secretion directed against all expressed HIV genes: relationship to rate of CD4 decline

Immune responses to human immunodeficiency virus (HIV) are detected at all stages of infection and are believed to be responsible for controlling viremia. This study seeks to determine whether gamma interferon (IFN-gamma)-secreting HIV-specific T-cell responses influence disease progression as defined by the rate of CD4 decline. The study population consisted of 31 subjects naive to antiretroviral therapy. All were monitored clinically for a median of 24 months after the time they were tested for HIV-specific responses. The rate of CD4+-T-cell loss was calculated for all participants from monthly CD4 counts. Within this population, 17 subjects were classified as typical progressors, 6 subjects were classified as fast progressors, and 8 subjects were classified as slow progressors. Peripheral blood mononuclear cells were screened for HIV-specific IFN-gamma responses to all expressed HIV genes. Among the detected immune responses, 48% of the recognized peptides were encoded by Gag and 19% were encoded by Nef gene products. Neither the breadth nor the magnitude of HIV-specific responses correlated with the viral load or rate of CD4 decline. The breadth and magnitude of HIV-specific responses did not differ significantly among typical, fast, and slow progressors. These results support the conclusion that although diverse HIV-specific IFN-gamma-secreting responses are mounted during the asymptomatic phase, these responses do not seem to modulate disease progression rates.