Mutations that suppress the thermosensitivity of green fluorescent protein

Background The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP has not always been straight forward. In particular, expression of GFP in cells that require incubation temperatures around 37°C has been problematic.Results We have carried out a screen for mutant forms of GFP that fluoresce more intensely than the wild-type protein when expressed in E. coli at 37°C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA act by preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can be manipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37°C.Conclusions The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells. The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, so it may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.     

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved β-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the β-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase. Received: 25 April 1997 / Accepted: 29 July 1997     

A new, rapid and reproducible method to obtain high quality endothelium in vitro

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.     

Ca2+-induced Ca2+ Release Phenomena in Mammalian Sympathetic Neurons Are Critically Dependent on the Rate of Rise of Trigger Ca2+

The role of ryanodine-sensitive intracellular Ca²⁺ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca²⁺ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca²⁺-mediated feedback loops, suggesting that it constitutes a Ca²⁺-induced Ca²⁺-release phenomenon. The dynamics of release is markedly affected when Sr²⁺ substitutes for Ca²⁺, indicating that Sr²⁺ release may operate with lower feedback gain than Ca²⁺ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca²⁺ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca²⁺ and Ca²⁺-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca²⁺ reservoir is full, the rapid initial Ca²⁺ rise determines a faster occupation of the ryanodine receptor Ca²⁺ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca²⁺ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca²⁺ content. This study suggests that transmembrane Ca²⁺ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca²⁺] near the Ca²⁺ release channels sufficiently fast to overcome their Ca²⁺-dependent inactivation. Conversely, caffeine-induced Ca²⁺ release can undergo considerable amplification especially when Ca²⁺ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca²⁺ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca²⁺ stores.     

Prepotent response inhibition and reaction times in children with attention deficit/hyperactivity disorder from a Caribbean community

Impairment in inhibitory control has been postulated as an underlying hallmark of attention deficit/hyperactivity disorder (ADHD), which can be utilized as a quantitative trait for genetic studies. Here, we evaluate whether inhibitory control, measured by simple automatized prepotent response (PR) inhibition variables, is a robust discriminant function for the diagnosis of ADHD in children and can be used as an endophenotype for future genetic studies. One hundred fifty-two school children (30.9% female, 67.8% with ADHD) were recruited. The ADHD checklist was used as the screening tool, whilst the DSM-IV Mini International Neuropsychiatry Interview, neurologic interview and neurologic examination, and the WISC III FSIQ test were administered as the gold standard procedure to assert ADHD diagnosis. A Go/No-Go task using a naturalistic and automatized visual signal was administered. A linear multifactor model (MANOVA) was fitted to compare groups including ADHD status, age, and gender as multiple independent factors. Linear discriminant analysis and the receiver operating characteristic curve were used to assess the predictive performance of PR inhibition variables for ADHD diagnosis. We found that four variables of prepotent response reaction time- and prepotent response inhibition established statistically significant differences between children with and without ADHD. Furthermore, these variables generated a strong discriminant function with a total classification capability of 73, 84% specificity, 68% sensitivity, and 90% positive predictive value for ADHD diagnosis, which support reaction times as a candidate endophenotype that could potentially be used in future ADHD genetic research.     

Increased Aβ production prompts the onset of glucose intolerance and insulin resistance

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD.     

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln⁰, a flightin null mutant, 2) Mhc¹³, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc⁶, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. Tension generation under isometric conditions was not impaired in fln⁰. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles. flight muscles; muscle mutants; myosin