IGHD II: A Novel GH-1 Gene Mutation (GH-L76P) Severely Affects GH Folding, Stability, and Secretion

CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.

Long Term Stability and Prediction of Soft Tissue Changes Following LeFort I Surgery

Submitted in partial fulfillment of the requirements for a Certificate in Orthodontics, Dept. of Orthodontics, University of Connecticut Health Center, 1991

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Stability and Transient Performance Assessment in a COTS-Module-Based Distributed DC/DC System

This paper introduces a method to analyze and predict stability and transient performance of a distributed system where COTS (Commercial-off-the-shelf) modules share an input filter. The presented procedure is based on the measured data from the input and output terminals of the power modules. The required information for the analysis is obtained by performing frequency response measurements for each converter. This attained data is utilized to compute special transfer functions, which partly determine the source and load interactions within the converters. The system level dynamic description is constructed based on the measured and computed transfer functions introducing cross-coupling mechanisms within the system. System stability can be studied based on the well-known impedance- related minor-loop gain at an arbitrary interface within the system.

General Report on Main Session 4: Embankments and dams, slope stability and landslides

The General Reporter presents the papers from the Authors, along with some personal contributions on the subjects discussed. Embankments are classified by their use. Different kinds of slope failure and remedial measures are dealt with, as well as investigations for material characterisation and selection.

Sensing colour stability and mixtures of powder paprika using optical reflectance and image analysis.

The production and industry of paprika present several problems related to quality and to production costs. One of the main difficulties is to obtain an objective and quick method for predicting quality. Quality in powder paprika involves: quantity of carotenoids and the appearance and stability of colour. The method used currently for determining quality is the measurement of absorbance at 460 nm wavelength, of an acetone extract of carotenoids, but there is no information about the appearance of the paprika or the stability of its colour with time. " Another important problem is the presence of mixtures of powdered paprika produced in the Spanish region of "La Vera", which has a peculiar way of production, with a high '' quality and price, with other products of lower quality. It is necessary to obtain methods which are able to detect the fraud.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Impedance-based stability and transient-performance assessment applying maximum peak criteria

The impedance-based stability-assessment method has turned out to be a very effective tool and its usage is rapidly growing in different applications ranging from the conventional interconnected dc/dc systems to the grid-connected renewable energy systems. The results are sometime given as a certain forbidden region in the complex plane out of which the impedance ratio--known as minor-loop gain--shall stay for ensuring robust stability. This letter discusses the circle-like forbidden region occupying minimum area in the complex plane, defined by applying maximum peak criteria, which is well-known theory in control engineering. The investigation shows that the circle-like forbidden region will ensure robust stability only if the impedance-based minor-loop gain is determined at the very input or output of each subsystem within the interconnected system. Experimental evidence is provided based on a small-scale dc/dc distributed system.

Axisymmetric long liquid bridges stability and resonances

In this paper mathematical expressions for minimum-volume stability limits and resonance frequencies of axisymmetric long liquid bridges are presented. These expressions are valid for a wide range of liquid bridge configurations, accounting for ef-fects like unequal disks and axial microgravity in the case of minimum-volume stability limits,and unequal disks, axial microgravity,non-zero viscosity and liquid bridge volume different from the cylindrical one in the case of resonance frequencies.

An experimental analysis od the stability and the dynamics of asixymmetric liquid bridges between unequal disks

In this paper experimental results related to both stability an the dynamic of a asiximmetric liquid bridge between unequal disk are presented. Experiments have been performed by using a drop tower facility and the response of the liquid bridge to a sudden change of the acceleration level acting on it has bee obtained.

Some aspects of lubrication in heavy regimes: thermal effects, stability and turbulence

In this work, a combination of numerical methods applied to thermohydrodynamic lubrication problems with cavitation is presented. It should be emphasized the difficulty of the nonlinear mathematical coupled model involving a free boundary problem, but also the simplicity of the algorithms employed to solve it. So, finite element discretizations for the hydrodynamic and thermal equations combined with upwind techniques for the convection terms and duality methods for nonlinear features are proposed. Additionally, a model describing the movement of the shaft is provided. Considering the shaft as a rigid body this model will consist of an ODE system relating acceleration of the center of gravity and external and pressure loads. The numerical experiments of mechanical stability try to clarify the position of the neutral stability curve. Finally, a rotating machine for ship propulsion involving both axial and radial bearings operating with nonconventional lubricants (seawater to avoid environmental pollution) is analyzed by using laminar and turbulent inertial flows.

Stability and dynamics in a hyperthermophilic protein with melting temperature close to 200°C

The rubredoxin protein from the hyperthermophilic archaebacterium Pyrococcus furiosus was examined by a hydrogen exchange method. Even though the protein does not exhibit reversible thermal unfolding, one can determine its stability parameters—free energy, enthalpy, entropy, and melting temperature—and also the distribution of stability throughout the protein, by using hydrogen exchange to measure the reversible cycling of the protein between native and unfolded states that occurs even under native conditions.

Expression, stability, and membrane integration of truncation mutants of bovine rhodopsin

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.