A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues. Received:17 December 1996 / Revised: 28 January 1997     

Is sleep apnea underdiagnosed in adult patients with osteogenesis imperfecta? –a single-center cross-sectional study

Background

Patients with Osteogenesis imperfecta (OI) suffer from increased bone fracture tendency generally caused by a mutation in genes coding for type I collagen. OI is also characterized by numerous co-morbidities, and recent data from questionnaire studies suggest that these may include increased risk for sleep apnea, a finding that lacks clinical evidence from cohort studies. In this cross-sectional study, 25 adults with OI underwent clinical otorhinolaryngology examination as well as overnight polysomnography to address the question. The participants were aged between 19 and 77?years, and ten of them had mild clinical OI phenotype, seven had a moderately severe phenotype, and eight had a severe phenotype.

Results

We found obstructive sleep apnea (apnea hypopnea index ≥5/h) in as many as 52% of the OI patients in the cohort. Unexpectedly, however, no correlation was present between sleep apnea and daytime sleepiness, experienced bodily pain, severity of OI, Mallampati score, or neck circumference.

Conclusions

Seeing that the usual predictors showed no association with occurrence of sleep apnea, we conclude that obstructive sleep apnea may easily be left as an undetected disorder in individuals with OI. Recurrent nocturnal hypoxia due to episodes of apneas can even affect bone metabolism, thereby further aggravating bone fragility in patients with OI.

     

The effect of trauma on dream content--A field study of Palestinian children.

In the present study, we compared the frequency and intensity of threatening events in the dreams of traumatized and nontraumatized Palestinian children. The aim of the study was to test some of the predictions and hypotheses derived from the Threat Simulation Theory proposing an evolutionary function for dreaming. Most, but not all, of our hypotheses were supported by the findings. We discuss the results in the light of the Threat Simulation Theory, and we also consider whether alternative theories of dream function are able to account for them. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development,Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning

The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a^+/-;P4ha1^+/- embryos demonstrated Wnt5a^-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.     

Effects of climate change and management on net climate impacts of production and utilization of energy biomass in Norway spruce with stable age‐class distribution

We studied the effects of climate change and forest management scenarios on net climate impacts (radiative forcing) of production and utilization of energy biomass, in a Norway spruce forest area over an 80‐year simulation period in Finnish boreal conditions. A stable age‐class distribution was used in model‐based analyses to identify purely the management effects under the current and changing climate (SRES B1 and A2 scenarios). The radiative forcing was calculated based on an integrated use of forest ecosystem model simulations and a life cycle assessment (LCA) tool. In this work, forest‐based energy was used to substitute coal, and current forest management (baseline management) was used as a reference management. In alternative management scenarios, the stocking was maintained 20% higher in thinning compared to the baseline management, and nitrogen fertilization was applied. Intensity of energy biomass harvest (e.g. logging residues, coarse roots and stumps) was varied in the final felling of the stands at the age of 80 years. Also, the economic profitability (NPV, 3% interest rate) of integrated production of timber and energy biomass was calculated for each management scenario. Our results showed that compared to the baseline management, climate benefits could be increased by maintaining higher stocking in thinning over rotation, using nitrogen fertilization and harvesting logging residues, stumps and coarse roots in the final felling. Under the gradually changing climate (in both SRES B1 and A2), the climate benefits were lower compared to the current climate. Trade‐offs between NPV and net climate impacts also existed.     

Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.     

Distinct germline progenitor subsets defined through Tsc2–mTORC1 signaling

Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional heterogeneity existing within this self-renewing population is poorly understood. Here, we define distinct subsets of undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation of mTORC1, a key growth-promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 throughout the SPC pool using Vasa-Cre promotes differentiation at the expense of self-renewal and leads to germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8-Cre did not compromise SPC function. SPC activity also appeared unaffected by Amh-Cre-mediated Tsc2 deletion within somatic cells of the niche. Importantly, we find that differentiation-prone SPCs have elevated mTORC1 activity when compared to SPCs with high self-renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with mTORC1-active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and defines phenotypically distinct SPC subpopulations with varying propensities for self-renewal and differentiation.     

Stochastic Dynamic Model for Estimation of Rate Constants and Their Variances from Noisy and Heterogeneous PET Measurements

Tissue heterogeneity, radioactive decay and measurement noise are the main error sources in compartmental modeling used to estimate the physiologic rate constants of various radiopharmaceuticals from a dynamic PET study. We introduce a new approach to this problem by modeling the tissue heterogeneity with random rate constants in compartment models. In addition, the Poisson nature of the radioactive decay is included as a Poisson random variable in the measurement equations. The estimation problem will be carried out using the maximum likelihood estimation. With this approach, we do not only get accurate mean estimates for the rate constants, but also estimates for tissue heterogeneity within the region of interest and other possibly unknown model parameters, e.g. instrument noise variance, as well. We also avoid the problem of the optimal weighting of the data related to the conventionally used weighted least-squares method. The new approach was tested with simulated time–activity curves from the conventional three compartment – three rate constants model with normally distributed rate constants and with a noise mixture of Poisson and normally distributed random variables. Our simulation results showed that this new model gave accurate estimates for the mean of the rate constants, the measurement noise parameter and also for the tissue heterogeneity, i.e. for the variance of the rate constants within the region of interest.     

Obesity history as a predictor of walking limitation at old age

Objective: To study whether walking limitation at old age is determined by obesity history. Research Methods and Procedures: In a retrospective longitudinal study based on a representative sample of the Finnish population of 55 years and older (2055 women and 1337 men), maximal walking speed, body mass, and body height were measured in a health examination. Walking limitation was defined as walking speed <1.2 m/s or difficulty in walking 0.5 km. Recalled height at 20 years of age and recalled weight at 20, 30, 40, and 50 years of age were recorded. Results: Subjects who had been obese at the age of 30, 40, or 50 years had almost a 4‐fold higher risk of walking limitation compared to non‐obese. Obesity duration increased the age‐ and gender‐adjusted risk of walking limitation among those who had been obese since the age of 50 (odds ratio, 4.33; 95% confidence interval, 2.59 to 7.23, n = 114), among the obese since the age of 40 [6.01 (2.55 to 14.14), n = 39], and among the obese since the age of 30 [8.97 (3.06 to 26.29), n = 14]. The risk remained elevated even among those who had previously been obese but lost weight during their midlife or late adulthood [3.15 (1.63 to 6.11), n = 71]. Discussion: Early onset of obesity and obesity duration increased the risk of walking limitation, and the effect was only partially mediated through current BMI and higher risk of obesity‐related diseases. Preventing excess weight gain throughout one's life course is an important goal in order to promote good health and functioning in older age.