Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3␤. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus͞hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases. Although many protein kinases phosphorylate tau at ADrelevant epitopes in vitro (reviewed in ref.2), only two have been copurified with microtubules, GSK3␤ and cdk5 (3, 4). To our knowledge, only these two kinases will phosphorylate tau in a cellular environment (e.g., refs. 5 and 6). We chose to focus on cdk5 because it is active predominantly in neurons whereas GSK3␤ plays a role in energy metabolism and is active in all cells. cdk5 is a member of the cyclin-dependent protein kinase gene family. Rather than cyclins, cdk5 associates with the positive allosteric regulators p35 (7), amino-terminal proteolytic fragments of p35 (e.g., p25; ref. 8), and p39 (9). These proteins share minimal amino acid sequence homology to cyclins, but the mechanism of activation of cdk5 by p25͞35 may be similar to the activation of cdk2 by cyclin A (10). p25͞35 is expressed predominantly in neurons, implying that most cdk5 activity is concentrated in neuronal structures (7,8). cdk5 plays a pivotal role in neuronal development as evidenced by the abnormal corticogenesis and perinatal lethality of cdk5 knockout mice (11) and the disturbances in neuronal migration and early death in p35 knockout mice (12). A number of potential cdk5 substrates have been identified and most are consistent with a putative role in neurite outgrowth and plasma membrane dynamics. These include cytoskeletal proteins such as tau and neurofilament (e.g., refs. 13 and 14) and synaptic vesicle proteins (15, 16). To clarify the potential role of cdk5 in neurodegenerative diseases in vivo, we overexpressed human p25 in the brains of transgenic mice to determine whether increased cdk5 activity would lead to hyperphosphorylation of tau and neurofilament and͞or cytoskeletal disturbances. Materials and MethodsAnimal Handling. All experimentation was performed under...

IntroductionProstaglandin E2 (PGE 2 ) is produced during inflammatory responses, and increased levels of PGE 2 help mediate some of the cardinal features of inflammation, including pain, edema, and fever (1, 2). PGE 2 exerts its effects through interactions with EP receptors, termed EP1-4 (3). Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting cyclooxygenase (COX) enzymes and thereby inhibiting prostaglandin production. In the context of this putative mechanism of action, direct cause-and-effect relationships between interruption of specific receptor-mediated signaling pathways and therapeutic actions have not been firmly established. While NSAIDs are effective analgesic agents, certain NSAIDs have a number of troublesome side effects that are due in part to their broad inhibition of a variety of COX products (4,5).Defining the molecular mechanisms underlying both the therapeutic and adverse actions of NSAIDs should provide useful targets for new, more specific therapeutic strategies. Therefore, we focused on a receptor for one of the prostaglandins (PGE 2 ), the EP1 receptor (6). We generated EP1-deficient mice by gene targeting and compared their physiological responses to genetically matched wild-type controls. We find that EP1 -/-animals have reduced nociceptive pain perception as well as altered cardiovascular homeostasis. These results demonstrate the critical actions of EP1 receptors in two physiological functions: pain perception and blood pressure regulation. Methods EP1 targeting vector construction and production of EP1 -/-mice.Mouse genomic clones containing Ptgerep1, mouse gene symbol for EP1 receptor, were isolated from a DBA/1lacJ genomic λ-phage library (Stratagene, La Jolla, California, USA). Long-template PCR was used to amplify 5′and 3′ fragments of the clone using T3 or T7 and EP1-specific primers. A 4.5-kb 5′ fragment and 6.0-kb 3′ fragment were cloned into pCRII vector (Invitrogen Corp., San Diego, California, USA). These fragments were sequence confirmed and subcloned into pHok, a plasmid containing PGK-neo and PGK-thymidine kinase cassettes. The EP1 targeting vector was designed to replace 671 bp of coding sequence with the PGK-neo cassette. This 671-bp coding region was Received for publication March 9, 1999, and accepted in revised form December 6, 2000.The lipid mediator prostaglandin E2 (PGE 2 ) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1 -/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1 -/-mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately ...

The use of quinolones in children and accumulation of data on the pharmacodynamics of these drugs have been limited and delayed by concern regarding their chondrotoxicity. A comprehensive review of the findings in animals compared with the cumulative published findings in children and adolescents (>7,000 to date) allows the conclusion that such concern is not justified. Prospective controlled studies in children are justifiable in view of a continuing lack of correlation between findings in juvenile animals and those in children and because of the selected therapeutic advantages of the current and newer quinolones.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ-or lesion-specific workshops planned by the ESTP.