ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.     

Identifying DNA splice sites using hypernetworks with artificial molecular evolution

Identifying DNA splice sites is a main task of gene hunting. We introduce the hyper-network architecture as a novel method for finding DNA splice sites. The hypernetwork architecture is a biologically inspired information processing system composed of networks of molecules forming cells, and a number of cells forming a tissue or organism. Its learning is based on molecular evolution. DNA examples taken from GenBank were translated into binary strings and fed into a hypernetwork for training. We performed experiments to explore the generalization performance of hypernetwork learning in this data set by two-fold cross validation. The hypernetwork generalization performance was comparable to well known classification algorithms. With the best hypernetwork obtained, including local information and heuristic rules, we built a system (HyperExon) to obtain splice site candidates. The HyperExon system outperformed leading splice recognition systems in the list of sequences tested.     

Brain Cytochrome Oxidase in Alzheimer''s Disease

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.     

Serotonin receptor expression in human prefrontal cortex: balancing excitation and inhibition across postnatal development

Serotonin and its receptors (HTRs) play critical roles in brain development and in the regulation of cognition, mood, and anxiety. HTRs are highly expressed in human prefrontal cortex and exert control over prefrontal excitability. The serotonin system is a key treatment target for several psychiatric disorders; however, the effectiveness of these drugs varies according to age. Despite strong evidence for developmental changes in prefrontal Htrs of rodents, the developmental regulation of HTR expression in human prefrontal cortex has not been examined. Using postmortem human prefrontal brain tissue from across postnatal life, we investigated the expression of key serotonin receptors with distinct inhibitory (HTR1A, HTR5A) and excitatory (HTR2A, HTR2C, HTR4, HTR6) effects on cortical neurons, including two receptors which appear to be expressed to a greater degree in inhibitory interneurons of cerebral cortex (HTR2C, HTR6). We found distinct developmental patterns of expression for each of these six HTRs, with profound changes in expression occurring early in postnatal development and also into adulthood. However, a collective look at these HTRs in terms of their likely neurophysiological effects and major cellular localization leads to a model that suggests developmental changes in expression of these individual HTRs may not perturb an overall balance between inhibitory and excitatory effects. Examining and understanding the healthy balance is critical to appreciate how abnormal expression of an individual HTR may create a window of vulnerability for the emergence of psychiatric illness.     

PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.