Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca²⁺ sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca²⁺-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca²⁺ binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca²⁺ affinity and thermal stability, whereas all the mutants showed decreased Ca²⁺ affinity and significantly lower thermal stability in both apo and Ca²⁺-loaded forms. No apparent cooperativity of Ca²⁺ binding was detected for any variant. Finally, the nonmyristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca²⁺ concentrations. Thus, we conclude that distorted Ca²⁺-sensor properties could lead to cone dysfunction.     

Missense mutations resulting in type 1 lissencephaly

Proper human brain formation is dependent upon the integrated activity of multiple genes. Malfunctioning of key proteins results in brain developmental abnormalities. Mutation(s) in the LIS1 gene or the X-linked gene doublecortin (DCX) results in a spectrum of disorders including lissencephaly, or smooth brain, and subcortical band heterotopia, or doublecortex. Here, we will focus on a particular subset of missense mutations in these two genes and their effect on protein structure and function.Received 4 August 2004; received after revision 26 September 2004; accepted 5 October 2004     

Lectin-induced lymphocyte stimulation in pigs — a kinetic study of NORs

Summary Early reactions of rRNA-cistrons in lectin-stimulated PBL cultures of domestic pigs are shown. Interphases showing 3 or more clusters of stained areas were attributed mainly to cells after first division. Clear differences are shown between PHA-and ConA-induced NOR-expression patterns compared to the patterns in PW-stimulated cultures.We thank Dr W. Gratze for valuable techniqual assistance, Dr M. Kundi from the Institute of Environmental Health of the University of Vienna for the statistical treatment of our data and the Ludwig Boltzmann Society for financial support.     

Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na⁺ channels and amplitude of Ca⁺⁺ currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.     

Amino acid absorption in jejunum and ileum in vivo — a kinetic comparison of function on surface area and regional bases

Summary Maximum absorptive capacities (V_max) for the jejunum and ileum corrected for the presence of an unstirred layer of water have been calculated for glycine, valine and methioninein vivo in fowls per unit surface area and per region. V_max per cm² showed that ileal enterocytes had a greater absorbing capacity than jejunal for glycine and valine but not for methionine. V_max for glycine and valine, calculated for the whole jejunum and ileum, however, were not different but for methionine the jejunal value was 1.9 times greater than the ileal.MAM is supported by an A.R.C. research grant (No. A50/37).     

CCN1/CYR61: the very model of a modern matricellular protein

CCN1 (CYR61) is a dynamically expressed, multifunctional matricellular protein that plays essential roles in cardiovascular development during embryogenesis, and regulates inflammation, wound healing and fibrogenesis in the adult. Aberrant CCN1 expression is associated with myriad pathologies, including various cancers and diseases associated with chronic inflammation. CCN1 promotes diverse and sometimes opposing cellular responses, which can be ascribed, as least in part, to disparate activities mediated through its direct binding to distinct integrins in different cell types and contexts. Accordingly, CCN1 promotes cell proliferation, survival and angiogenesis by binding to integrin α_vβ₃, and induces apoptosis and senescence through integrin α₆β₁ and heparan sulfate proteoglycans. The ability of CCN1 to trigger the accumulation of a robust and sustained level of reactive oxygen species underlies some of its unique activities as a matrix cell-adhesion molecule. Emerging studies suggest that CCN1 might be useful as a biomarker or therapeutic target in certain diseases.     

Prediction in a duration model

Predictors of mean duration and an arbitrary quantile are given for the Weibull regression model for duration data. Associated prediction variances arising from maximum likelihood and least squares estimation are given. In an empirical example, based on duration of employment data, the uses of various model diagnostics and the predictors are illustrated.     

RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.     

Myo1c mutations associated with hearing loss cause defects in the interaction with nucleotide and actin

Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide and actin using a truncated construct, Myo1c^1IQ-SAH, which includes an artificial lever arm. Results indicate that mutation R156W, near switch 1, affects the nucleotide-binding pocket and the calcium binding by disrupting switch 1 movement. Mutation V252A, in the K helix of the upper 50 kDa domain, showed reduced actin affinity consistent with disruption of communication between the actin- and nucleotide-binding sites. T380M, in a Myo1c-specific insert in the HO linker, displayed aberrant changes in most kinetic parameters and uncoupling of the ATPase from motility. These data allow for an interpretation of how these mutations might affect adaptation.     

A predictive model of train delays on a railway line

Delay prediction is an important issue associated with train timetabling and dispatching. Based on real-world operation records, accurate forecasting of delays is of immense significance in train operation and decisions of dispatchers. In this study, we established a model that illustrates the interaction between train delays and their affecting factors via train describer records on a Dutch railway line. Based on the main factors that affect train delay and the time series trend, we determined the independent and dependent variables. A long short-term memory (LSTM) prediction model in which the actual delay time corresponded to the dependent variable was established via Python. Finally, the prediction accuracy of the random forest model and artificial neural network model was compared. The results indicated that the LSTM model outperformed the other two models.     

A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9 weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.