Endogenous monoamine oxidase inhibitory activity and HPA activation in the pig

Previously we have shown that an increase in endogenous monoamine oxidase A inhibitory activity (MAO-AI), measured in human saliva, both precedes and predicts psychological stress-induced activation of the hypothalamic pituitary adrenal (HPA) axis, as determined by the cortisol response. We now report the relationship between endogenous MAO-AI and the cortisol response in the plasma of prepubertal pigs (n=5 or 6) under two experimental paradigms of HPA activation. In the first condition, pigs were physically restrained (snaring) for 15 minutes. Blood samples were taken from indwelling catheters at intervals before and after snaring (a sampling period of about 1 hour), and at the same time intervals on a separate day to provide baseline measures. Both cortisol concentration and percentage MAO-AI were determined in each plasma sample. There was a pronounced cortisol response on the snaring day (cortisol peaked 30 minutes after the start of the snaring). There was also a significant MAO-AI response to snaring which peaked 15 minutes after the start of the stress challenge. In the second experimental paradigm, bacterial endotoxin (LPS: 20microg/pig) was used to induce HPA activation and plasma cortisol and MAO-AI were determined. This time, however, the cortisol response was not preceded by any change in MAO-AI. We conclude that generation of MAO-AI, which is associated with HPA activation induced by psychological stress, is not a component of the pathways involved in immunological stimulation of the HPA axis.     

Extracellular N-acetylaspartate depletion in traumatic brain injury

N-Acetylaspartate (NAA) is almost exclusively localized in neurons in the adult brain and is present in high concentration in the CNS. It can be measured by proton magnetic resonance spectroscopy and is seen as a marker of neuronal damage and death. NMR spectroscopy and animal models have shown NAA depletion to occur in various types of chronic and acute brain injury. We investigated 19 patients with traumatic brain injury (TBI). Microdialysis was utilized to recover NAA, lactate, pyruvate, glycerol and glutamate, at 12-h intervals. These markers were correlated with survival and a 6-month Glasgow Outcome Score. Eleven patients died and eight survived. A linear mixed model analysis showed a significant effect of outcome and of the interaction between time of injury and outcome on NAA levels (p = 0.009 and p = 0.004, respectively). Overall, extracellular NAA was 34% lower in non-survivors. A significant non-recoverable fall was observed in this group from day 4 onwards, with a concomitant rise in lactate-pyruvate ratio and glycerol. These results suggest that mitochondrial dysfunction is a significant contributor to poor outcome following TBI and propose extracellular NAA as a potential marker for monitoring interventions aimed at preserving mitochondrial function.     

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid

The purpose of these experiments was to examine the effects of dietary antioxidant supplementation with vitamin E (VE) and alpha-lipoic acid (alpha-LA) on biochemical and physiological responses to in vivo myocardial ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo old) were assigned to either 1) a control diet (CON) or 2) a VE and alpha-LA supplemented diet (ANTIOX). After a 14-wk feeding period, animals in each group underwent an in vivo I-R protocol (25 min of myocardial ischemia and 15 min of reperfusion). During reperfusion, peak arterial pressure was significantly higher (P < 0.05) in ANTIOX animals compared with CON diet animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in ANTIOX animals. Compared with ANTIOX animals, heart homogenates from CON animals experienced significantly less (P < 0.05) oxidative damage when exposed to five different in vitro radical producing systems. These data indicate that dietary supplementation with VE and alpha-LA protects the aged rat heart from I-R-induced lipid peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.     

Regulation of Mg2+-independent Ca2+-ATPase by a low molecular mass protein purified from bovine brain

The goat sperm microsomal membranes have been found to contain an Mg2+-independent Ca2+-ATPase, a low affinity but highly active enzyme sharing similarities with the SERCA family of ATPases. The present study reports the identification and characterization of a 14 kilodalton cytosolic protein from bovine brain which can act as an endogenous stimulator of the enzyme with an S50 (concentration producing 50% stimulation) of 0.8 mu molar. Kinetic analysis suggests that the stimulation is noncompetitive with respect to the substrate, and the binding site(s) of the stimulator and substrate are distinct. Binding of the stimulator to the enzyme is reversible. The stimulator increases the affinity of the enzyme for calcium as evident from a decrease in K0.5 of the enzyme for calcium in presence of the stimulator. Radioactive labeling of the enzyme with [gamma-32P]-ATP suggests that the stimulator enhances the rate of dephosphorylation of the phosphoenzyme intermediate without altering the phosphorylation reaction step. The stimulatory effect of the protein has been observed only for the Mg2+-independent form of the enzyme, the Mg2+-dependent form being unaffected.     

Quantitative trait locus study design from an information perspective

We examine the efficiency of different genotyping and phenotyping strategies in inbred line crosses from an information perspective. This provides a mathematical framework for the statistical aspects of QTL experimental design, while guiding our intuition. Our central result is a simple formula that quantifies the fraction of missing information of any genotyping strategy in a backcross. It includes the special case of selectively genotyping only the phenotypic extreme individuals. The formula is a function of the square of the phenotype and the uncertainty in our knowledge of the genotypes at a locus. This result is used to answer a variety of questions. First, we examine the cost-information trade-off varying the density of markers and the proportion of extreme phenotypic individuals genotyped. Then we evaluate the information content of selective phenotyping designs and the impact of measurement error in phenotyping. A simple formula quantifies the information content of any combined phenotyping and genotyping design. We extend our results to cover multigenotype crosses, such as the F(2) intercross, and multiple QTL models. We find that when the QTL effect is small, any contrast in a multigenotype cross benefits from selective genotyping in the same manner as in a backcross. The benefit remains in the presence of a second unlinked QTL with small effect (explaining <20% of the variance), but diminishes if the second QTL has a large effect. Software for performing power calculations for backcross and F(2) intercross incorporating selective genotyping and marker spacing is available from http://www.biostat.ucsf.edu/sen.     

A Study of the Mechanism of the Chaperone-like Function of an scFv of Human Creatine Kinase by Computer Simulation

A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies'' chaperone-like function.     

Potential-dependent calcium channels in rabbit jejunum

Potential-dependent calcium channels have been studied in the isolated rabbit jejunum. A biphasic response was observed, a transient and rapid contraction followed by partial relaxation and a sustained contraction attaining a plateau state. These are similar to the phasic and tonic responses observed in the isolated smooth muscles of other species. Both the responses are susceptible to blockade by the calcium antagonists bepridil, diltiazem, nifedipine, PN 200-110 (isradipine), and verapamil. Two calcium pools or the presence of two channels affecting the two responses are proposed.