Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Proteomic analysis of mouse brain cortex identifies metabolic down-regulation as a general feature of ischemic pre-conditioning

J. Neurochem. (2012) 122, 1219-1229. ABSTRACT: The molecular mechanisms that lead to ischemic pre-conditioning are not completely understood, and proteins are important players. We compared the mouse brain cortex proteome from different ischemia sets: transient (7?min) middle cerebral artery occlusion (7'MCAo, pre-conditioning stimulus), permanent MCAo (pMCAo, severe ischemia), and pMCAo 4?days after 7'MCAo (7'MCAo/pMCAo, pre-conditioned model). Proteins were analyzed by two-dimensional electrophoresis coupled to liquid chromatography-tandem mass spectrometry. Overall, 28 proteins were expressed differentially from sham controls, and identified. The ischemic pre-conditioning stimulus alone up-regulated the stress protein heat-shock protein 70 (HSP70), possibly activated by the androgen receptor. Western blotting confirmed the increased expression of HSP70 and showed that androgen receptor expression paralleled that of HSP70. In the ischemic-tolerant group (7'MCAo/pMCAo), a number of proteins over-expressed after pMCAo returned to sham levels, seven proteins remained up-regulated as in pMCAo, and five proteins mainly involved in energy metabolism and mitochondrial electron transport and unchanged in pMCAo were down-regulated only in ischemic tolerance, suggesting a role in brain pre-conditioning. Astrocytes participated in ischemic-tolerance induction, as shown by the down-regulation of glutamine synthetase in the 7'MCAo/pMCAo group. The results suggest that metabolic down-regulation was a general feature of ischemic pre-conditioning, playing a pivotal role in neuroprotection.     

Ultrastructural changes in the interstitial cells of Cajal and gastric dysrhythmias in mice lacking full-length dystrophin (mdx mice)

At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca(2+) handling, and with defective generation and regeneration of slow wave activity.     

Inhibition of growth hormone secretion in mild primary hyperparathyroidism

INTRODUCTION: Impairment in growth hormone (GH) secretion has been reported to occur in primary hyperparathyroidism (PHP) with strikingly elevated (>150 pg/ml) plasma PTH and free Ca levels. Patients with these characteristics are relatively few, whereas the great majority of patients with biochemically diagnosed PHP are asymptomatic and show borderline or slightly elevated plasma PTH and Ca levels. We wondered whether also patients in these latter conditions show a defective GH secretory pattern. METHODS: In order to answer this question, 8 female subjects (mean age +/- SE: 44 +/- 1.3 years) were selected at the time of a checkup examination from a larger population of persons in fairly good clinical condition. Inclusion criteria were plasma PTH values slightly above the normal range (up to 50% higher than the maximum limit) with free Ca levels in the upper normal range or slightly higher (experimental group). Normal values in our laboratory are ionized calcium: 1.22-1.42 mmol/ml and plasma PTH: 12-72 pg/ml. A group of 15 age-matched healthy women with plasma PTH and Ca levels in the middle normal range and significantly lower than values found in the experimental group was also selected and used as control. Experimental and control groups were tested with arginine [0.5 mg/kg body weight (BW)] infused intravenously over 30 min and arginine plus GH-releasing hormone (GHRH; 1 microg/kg BW in an intravenous bolus injection). The GH responses to these challenging stimulations were compared between groups. RESULTS: Basal serum GH values were similar in all subjects. Both arginine and arginine plus GHRH induced a significant GH rise in both groups; however, the GH responses were significantly lower in the experimental than in the control group. Mean GH peak was 27.7 and 14.6 times higher than baseline after arginine and 57.5 and 26.6 times higher than baseline after arginine plus GHRH in the control and experimental group, respectively. No significant correlation was observed between PTH or Ca levels and the GH responses to challenging stimuli in any group. CONCLUSION: These data show that impairment in GH secretion is associated with slightly elevated levels of PTH in the presence of serum Ca values in the upper normal range. GH responses to stimulations were reduced by about 50% in our hyperparathyroid subjects. A long-time duration of this relatively small decline of GH secretory activity may be supposed to contribute to age-related catabolic processes in a large number of patients with mild primary hyperparathyroidism.     

Variation of trabecular architecture in proximal femur of postmenopausal women

This investigation of microstructure in the human proximal femur probes the relationship between the parameters of the FRAX index of fracture risk and the parameters of bone microstructure. The specificity of fracture sites at the proximal femur raises the question of whether trabecular parameters are site-specific during post-menopause, before occurrence of fragility fracture. The donated proximal femurs of sixteen post-menopausal women in the sixth and seventh decades of life, free of metabolic pathologies and therapeutic interventions that could have altered the bone tissue, constituted the material of the study. We assessed bone mineral density of the proximal femurs by dual energy X-ray absorptiometry and then sectioned the femurs through the center of the femoral head and along the femoral neck axis. For each proximal femur, morphometry of trabeculae was conducted on the plane of the section divided into conventional regions and sub-regions consistent with the previously identified trabecular families that provide regions of relatively homogeneous microstructure. Mean trabecular width and percent bone area were calculated at such sites. Our findings indicate that each of mean trabecular width and percent bone area vary within each proximal femur independently from each other, with dependence on site. Both trabecular parameters show significant differences between pairs of sites. We speculate that a high FRAX index at the hip corresponds to a reduced percent bone area among sites that gives a more homogeneous and less site-specific quality to the proximal femur. This phenomenon may render the local tissue less able to carry out the expected mechanical function.     

Hyperlipidemia affects multiscale structure and strength of murine femur

To improve bone strength prediction beyond limitations of assessment founded solely on the bone mineral component, we investigated the effect of hyperlipidemia, present in more than 40% of osteoporotic patients, on multiscale structure of murine bone. Our overarching purpose is to estimate bone strength accurately, to facilitate mitigating fracture morbidity and mortality in patients. Because (i) orientation of collagen type I affects, independently of degree of mineralization, cortical bone?s micro-structural strength; and, (ii) hyperlipidemia affects collagen orientation and μCT volumetric tissue mineral density (vTMD) in murine cortical bone, we have constructed the first multiscale finite element (mFE), mouse-specific femoral model to study the effect of collagen orientation and vTMD on strength in Ldlr^−/−, a mouse model of hyperlipidemia, and its control wild type, on either high fat diet or normal diet. Each µCT scan-based mFE model included either element-specific elastic orthotropic properties calculated from collagen orientation and vTMD (collagen-density model) by experimentally validated formulation, or usual element-specific elastic isotropic material properties dependent on vTMD-only (density-only model). We found that collagen orientation, assessed by circularly polarized light and confocal microscopies, and vTMD, differed among groups and that microindentation results strongly correlate with elastic modulus of collagen-density models (r²=0.85, p=10⁻⁵). Collagen-density models yielded (1) larger strains, and therefore lower strength, in simulations of 3-point bending and physiological loading; and (2) higher correlation between mFE-predicted strength and 3-point bending experimental strength, than density-only models. This novel method supports ongoing translational research to achieve the as yet elusive goal of accurate bone strength prediction.