Untangling genetic networks of panic,phobia, fear and anxiety

As is the case for normal individual variation in anxiety levels, the conditions panic disorder, agoraphobia and other phobias have a significant genetic basis. Recent reports have started to untangle the genetic relationships between predispositions to anxiety and anxiety disorders.     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Role of thromboxane in producing portal hypertension following trauma-hemorrhage

Thromboxane A2 (TXA2) and endothelin-1 (ET-1) have been proposed as the important vasoconstrictors that increase portal venous resistance in paracrine or autocrine fashion. We hypothesized that the hepatic damage following trauma-hemorrhage (T-H) is induced by the impaired hepatic circulation due to the increased production of vasoconstrictors such as ET-1 and TXA2 by the liver. To test this, male Sprague-Dawley rats (n = 6/group) were subjected to trauma (i.e., midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. At 2 or 5 h after the end of resuscitation, the liver was isolated and perfused and portal inflow pressure, bile flow, and release of ET-1 and thromboxane B2 (TXB2; a stable metabolite of TXA2) into the perfusate were measured. The level of portal pressure was higher at 5 h following T-H compared with 2 h after T-H and sham. The portal pressure was inversely correlated to the amount of bile production. Furthermore, the bile flow was significantly correlated to the hepatic damage as evidenced by release of lactate dehydrogenase into the perfusate. The level of ET-1 at 5 h following T-H in the perfusate after 30 min of recirculation did not show any difference from sham. However, the levels of TXB2 in the T-H group were significantly higher than those in sham at that interval. These results indicate that the increased release of TXA2 but not ET-1 following T-H might be responsible for producing the increased portal resistance, decreased bile production, and hepatic damage.     

Increased thermoregulatory responsiveness in cold adapted but not in hyperthyroid hypermetabolic rats

Cold-adapted (CA) rats, unlike non-adapted (NA) ones, give exaggerated metabolic response to acute cold exposure, with paradoxical "overshoot" core temperature (Tc) rise in the cold, and they also give enhanced hyperthermia to central injection of prostaglandin E1 (PGE1). The adaptation-dependent differences might be explained either by the high thermogenic capacity of peripheral tissues in CA rats or by differences in the central processing of regulatory signals. If high tissue metabolism sufficiently explains the extreme responses of CA animals, other hypermetabolic states (with high resting metabolic rate, RMR), e.g. hyperthyroidism, should also be accompanied by enhanced reactions. In the present study thermoregulatory responses to acute cold exposure or to PGE1 were compared in hypermetabolic CA, similarly hypermetabolic thyroxine-treated (T4) and control non-hypermetabolic NA rats (mean RMR = 8.12, 8.47 and 6.03 W kg(-1), respectively). Cold exposure was followed by paradoxical core temperature (Tc) rise of 0.5 to 0.7 degrees C only in CA rats, but by Tc fall (0.8 to 2.1 degrees C) in NA and T4 animals. Identical central stimuli (PGE1) induced larger elevations of Tc and metabolic rate in CA rats than in similarly hypermetabolic T4 or in non-hypermetabolic NA animals (mean Tc rise of 1.9 degrees C in CA vs. 0.9 degrees C in T4 and 1.0 degrees C in NA rats). Vasodilatation thresholds were also similar in NA and T4, but lowered in CA animals. A hypermetabolic status, per se, does not seem to explain the enhanced thermoregulatory responsiveness of CA animals, adaptation-induced central regulatory changes may be more important for the "overshoot" phenomenon.     

Akt activation induced by an antioxidant compound during ischemia-reperfusion

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.     

In-vitro germination and growth of lily pollen tubes is affected by protein phosphatase inhibitors

Inhibitors of type-2A protein phosphatase (PPase-2A), calyculin A (cal A) and okadaic acid (OA), inhibit pollen grain germination and growth of pollen tubes of Lilium longiflorum Thunb. at nanomolar concentrations. Half-maximal inhibition of cytoplasmic PPase-2A activity was below 0.1 nM for cal A and at 0.7 nM for OA. Other protein phosphatase inhibitors (tautomycin, cypermethrin, and dephostatin) were less effective. The OA- and cal A-sensitive as well as dephostatin-sensitive PPase activity in the cytoplasm did not change during germination and growth of pollen tubes. Addition of cal A and OA disturbed the direction of pollen tube growth and the distribution of cytoplasmic organelles and caused cell wall thickenings as observed by light and electron microscopy. Inhibition of PPase-2A caused multiple effects at the cellular level, cytoskeletal elements being a putative target of PPase-2A activity. Received: 30 March 1998 / Accepted: 6 July 1998     

Photosynthesis of lichens from lichen-dominated communities in the alpine/nival belt of the Alps – II: Laboratory and field measurements of CO2 exchange and water relations

CO₂ exchange and water relations of selected lichen species were investigated in the field and also in the laboratory, at a height of 3106 m above sea level in the Austrian Alps, during the short snowless summer period from middle of July to the end of August. In the course of the field investigations, clear summer days were quite rare. Altogether 14 diurnal courses of CO₂ exchange were measured spanning a time of 255 h of measurements.The air temperatures measured close to the ground ranged between −0.7 and 17.1 °C and their daily fluctuation was lower than 10.7 °C. Fog was present for more than one-third of the measuring period and relative humidity (RH) exceeded 90% in almost half of the time. Temperature optimum of net photosynthesis (NP) of Xanthoria elegans and Brodoa atrofusca determined in the laboratory increased with increasing photosynthetic photon flux density (PPFD) from 1.5 to 11.3 °C and the maximal CO₂ uptake was found to be at 10 °C. In the field the lichens were metabolically active at air temperatures between −0.7 and 12.8 °C. The light compensation points (LCP) of both lichen species ranged in the laboratory between 50 and 200 μmol m⁻² s⁻¹ PPFD (0–20 °C) and in the field between 22 and 56 μmol m⁻² s⁻¹ PPFD (3–8 °C). At 30 °C the NP of X. elegans surpassed the LCP, whereas B. atrofusca remained below the LCP. NP in X. elegans did not reach light saturation at 1500 μmol m⁻² s⁻¹ PPFD. NP in B. atrofusca reached light saturation at low temperatures (−5 to +5 °C). At higher temperatures light saturation was almost detectable. On sunny days the lichens in the field were metabolically active only for 3 h during the early morning. In this time they reached the maximal values or values close to their maximal CO₂ uptake in situ. Under dry weather conditions the lichens dried out to a minimal water content (WC) of 5–12% which is below the moisture compensation point (MCP) of 34–25%. The optimal WC was between 90% and 120% dry weight (DW) in B. atrofusca and Umbilicaria cylindrica, in X. elegans between 140% and 180% DW. Species specific differences in water-holding capacity, desiccation intensity and in the compensation points of temperature, light and moisture are responsible for differences in metabolic activity. The lichens were active during less than half of the observation time. Total time of NP of X. elegans was 24% of the measuring period, for U. cylindrica 22% and for B. atrofusca 16%.     

Pre-Analytical Sample Quality: Metabolite Ratios as an Intrinsic Marker for Prolonged Room Temperature Exposure of Serum Samples

Advances in the “omics” field bring about the need for a high number of good quality samples. Many omics studies take advantage of biobanked samples to meet this need. Most of the laboratory errors occur in the pre-analytical phase. Therefore evidence-based standard operating procedures for the pre-analytical phase as well as markers to distinguish between ‘good’ and ‘bad’ quality samples taking into account the desired downstream analysis are urgently needed. We studied concentration changes of metabolites in serum samples due to pre-storage handling conditions as well as due to repeated freeze-thaw cycles. We collected fasting serum samples and subjected aliquots to up to four freeze-thaw cycles and to pre-storage handling delays of 12, 24 and 36 hours at room temperature (RT) and on wet and dry ice. For each treated aliquot, we quantified 127 metabolites through a targeted metabolomics approach. We found a clear signature of degradation in samples kept at RT. Storage on wet ice led to less pronounced concentration changes. 24 metabolites showed significant concentration changes at RT. In 22 of these, changes were already visible after only 12 hours of storage delay. Especially pronounced were increases in lysophosphatidylcholines and decreases in phosphatidylcholines. We showed that the ratio between the concentrations of these molecule classes could serve as a measure to distinguish between ‘good’ and ‘bad’ quality samples in our study. In contrast, we found quite stable metabolite concentrations during up to four freeze-thaw cycles. We concluded that pre-analytical RT handling of serum samples should be strictly avoided and serum samples should always be handled on wet ice or in cooling devices after centrifugation. Moreover, serum samples should be frozen at or below -80°C as soon as possible after centrifugation.     

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics,Spindle Orientation and Proper Chromosome Segregation during Mitosis

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.