Augmented Hypothalamic Corticotrophin-Releasing Hormone mRNA and Corticosterone Responses to Stress in Adult Rats Exposed to Perinatal Hypoxia

Stressful events before or just after parturition alter the subsequent phenotypical response to stress in a general process termed programming. Hypoxia during the period before and during parturition, and in the postnatal period, is one of the most common causes of perinatal distress, morbidity, and mortality. We have found that perinatal hypoxia (prenatal day 19 to postnatal day 14) augmented the corticosterone response to stress and increased basal corticotrophin-releasing hormone (CRH) mRNA levels in the parvocellular portion of the paraventricular nucleus (PVN) in 6-month-old rats. There was no effect on the levels of hypothalamic parvocellular PVN vasopressin mRNA, anterior pituitary pro-opiomelanocortin or CRH receptor-1 mRNA, or hippocampus glucocorticoid receptor mRNA. We conclude that hypoxia spanning the period just before and for several weeks after parturition programmes the hypothalamic-pituitary-adrenal axis to hyper-respond to acute stress in adulthood, probably as a result of drive from the parvocellular CRH neurones

Imaging the mitochondrial permeability transition in single cells: Free radicals and calcium as determinants of cell fate

The textbook interpretation of mitochondrial function has had to be revised in the light of recent discoveries establishing a central role for mitochondria in modulating cell signalling. In particular, the opening of the mitochondrial permeability transition pore (MPTP), a large conductance pore in the tightly impermeable inner mitochondrial membrane, may determine cell fate by inducing either necrotic or apoptotic cell death. I have developed an experimental protocol in which MPTP opening can be reliably induced in living cells. I have then used this as a model to study the relative roles of calcium and reactive oxygen species (ROS) in permeability transition and explored the consequences of these processes for cell fate. The membrane-permeant, fluorescent dye, tetramethylrhodamine ethyl ester (TMRE), accumulates in mitochondria where its fluorescence signals changes in mitochondrial transmembrane potential ( m). Digital imaging of adult rat cortical astrocytes loaded with TMRE revealed spontaneous, transient depolarisations of individual mitochondria leading to a gradual and complete mitochondrial depolarisation in whole cells. Single mitochondria could depolarise and repolarise several times. Treatment of the cells with cyclosporin (an inhibitor of pore opening) significantly reduced the depolarisations suggesting that they were caused by MPTP opening and that TMRE could be used to signal MPTP in intact single cells. This model of MPTP opening was used to study the underlying mechanisms involved in MPTP opening. Illumination of TMRE produces ROS. Attenuation of the excitation light intensity or treatment with free radical scavengers reduced the frequency of the depolarisations, confirming a role for ROS in this model of permeability transition. Mitochondrial calcium loading is thought to be a prerequisite for permeability transition and I found that chelation of intracellular calcium or depletion of endoplasmic reticulum calcium stores inhibited the transient openings. Thus either free radical scavengers or chelation of calcium inhibited pore opening in this model, suggesting an interplay between ROS production and mitochondrial calcium loading in permeability transition, perhaps as a result of ROS-induced calcium release from intracellular stores. The fate of cells that were illuminated with low intensity light, where MPTP was transient, was similar to non-illuminated controls, suggesting that opening of the pore does not inevitably lead to cell death. Those cells in which collapse of [delta][psi]m was complete, however, died by a necrotic pathway

Predicted structures of agonist and antagonist bound complexes of adenosine A_3 receptor

We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the four subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A_3 adenosine receptor (hAA_3R) for subtype selective agonists and antagonists. We found that all four A_3 agonists stabilize the 15th lowest conformation of apo-hAA_3R while also binding strongly to the 1st and 3rd. In contrast the four A_3 antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA_3R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We found that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA_3R shows favorable interactions to three subtype variable residues, I253^(6.58), V169^(EL2), and Q167^(EL2), while the predicted structure for hAA_(2A)R shows weakened to the corresponding amino acids: T256^(6.58), E169^(EL2), and L167^(EL2), explaining the observed subtype selectivity

Iterative explicit guidance for low thrust spacecraft.

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77340/1/AIAA-1972-916-170.pd