Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Detection of 14-3-3zeta in cerebrospinal fluid following experimentally evoked seizures

Surrogate and peripheral (bio)markers of neuronal injury may be of value in assessing effects of seizures on the brain or epilepsy development following trauma. The presence of 14-3-3 isoforms in cerebrospinal fluid (CSF) is a diagnostic indicator of Creutzfeldt-Jakob disease but these proteins may also be present following acute neurological insults. Here, we examined neuronal and 14-3-3 proteins in CSF from rats after seizures. Seizures induced by intra-amygdala microinjection of 0.1 microg kainic acid (KA) caused damage which was mainly restricted to the ipsilateral CA3 subfield of the hippocampus. 14-3-3zeta was detected at significant levels in CSF sampled 4 h after seizures compared with near absence in control CSF. Neuron-specific nuclear protein (NeuN) was also elevated in CSF in seizure rats. CSF 14-3-3zeta levels were significantly lower in rats treated with 0.01 microg KA. These data suggest the presence of 14-3-3zeta within CSF may be a biomarker of acute seizure damage.     

Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3gamma and -zeta were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3gamma levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3zeta, while cytoplasm-localized 14-3-3beta, -epsilon and -eta remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3epsilon and -zeta levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures.     

Loss of p53 results in protracted electrographic seizures and development of an aggravated epileptic phenotype following status epilepticus

The p53 tumor suppressor is a multifunctional protein, which regulates cell cycle, differentiation, DNA repair and apoptosis. Experimental seizures up-regulate p53 in the brain, and acute seizure-induced neuronal death can be reduced by genetic deletion or pharmacologic inhibition of p53. However, few long-term functional consequences of p53 deficiency have been explored. Here, we investigated the development of epilepsy triggered by status epilepticus in wild-type and p53-deficient mice. Analysis of electroencephalogram (EEG) recordings during status epilepticus induced by intra-amygdala kainic acid (KA) showed that seizures lasted significantly longer in p53-deficient mice compared with wild-type animals. Nevertheless, neuronal death in the hippocampal CA3 subfield and the neocortex was significantly reduced at 72 h in p53-deficient mice. Long-term continuous EEG telemetry recordings after status epilepticus determined that the sum duration of spontaneous seizures was significantly longer in p53-deficient compared with wild-type mice. Hippocampal damage and neuropeptide Y distribution at the end of chronic recordings was found to be similar between p53-deficient and wild-type mice. The present study identifies protracted KA-induced electrographic status as a novel outcome of p53 deficiency and shows that the absence of p53 leads to an exacerbated epileptic phenotype. Accordingly, targeting p53 to protect against status epilepticus or related neurologic insults may be offset by deleterious consequences of reduced p53 function during epileptogenesis or in chronic epilepsy.     

The Genetic Architecture of Climatic Adaptation of Tropical Cattle

Adaptation of global food systems to climate change is essential to feed the world. Tropical cattle production, a mainstay of profitability for farmers in the developing world, is dominated by heat, lack of water, poor quality feedstuffs, parasites, and tropical diseases. In these systems European cattle suffer significant stock loss, and the cross breeding of taurine x indicine cattle is unpredictable due to the dilution of adaptation to heat and tropical diseases. We explored the genetic architecture of ten traits of tropical cattle production using genome wide association studies of 4,662 animals varying from 0% to 100% indicine. We show that nine of the ten have genetic architectures that include genes of major effect, and in one case, a single location that accounted for more than 71% of the genetic variation. One genetic region in particular had effects on parasite resistance, yearling weight, body condition score, coat colour and penile sheath score. This region, extending 20 Mb on BTA5, appeared to be under genetic selection possibly through maintenance of haplotypes by breeders. We found that the amount of genetic variation and the genetic correlations between traits did not depend upon the degree of indicine content in the animals. Climate change is expected to expand some conditions of the tropics to more temperate environments, which may impact negatively on global livestock health and production. Our results point to several important genes that have large effects on adaptation that could be introduced into more temperate cattle without detrimental effects on productivity.     

Genetic Pleiotropy for Resting Metabolic Rate with Fat-Free Mass and Fat Mass: The Québec Family Study

Shared genetic and familial environmental causes for the associations among resting metabolic rate (RMR), fat-free mass (FFM), and fat mass (FM) were investigated in families participating in phase 2 of the Québec Family Study. A multivariate familial correlation model assessing the pattern of significant cross-trait correlations between family members (e.g., RMR in parents with FFM in offspring) was used to infer the etiology of the associations. For each of FM and FFM with RMR, significant sibling, parent-offspring, and intraindividual cross-trait correlations suggest the associations are familial. Furthermore, the lack of significant spouse cross-trait correlations suggests that the familial aggregation is primarily genetic. Bivariate heritability estimates suggest that as much as 45% to 50% of the shared variance between FFM and RMR may be genetic, and as much as 28% to 34% for FM and RMR. This study supports the notion that the gene(s) affecting each of FFM and FM also influence the RMR. Moreover, the lack of any familial associations between FFM and FM suggests that the effects of each body size component on RMR are independent, i.e., more than one genetic source on the RMR-body size association. The possibility that RMR is an oligogenic trait (i.e., more than one underlying genetic etiology) should be further investigated using more complex multivariate segregation methods until specific genes can be tested.     

Common activation of canonical Wnt signaling in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.     

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ-amino butyric acid-mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligand-gated P2X receptors. Here we report that SE induced by intra-amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure-induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure-suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure-responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.