Living in the cold: freeze-induced gene responses in freeze-tolerant vertebrates

1. Winter survival for numerous cold-blooded animals includes freeze tolerance: the ability to endure the conversion of as much as 65% of total body water into extracellular ice. Selected molecular adaptations underlying freeze tolerance (e.g. cryoprotectants, ice nucleating proteins) have been widely studied, but the full range of metabolic adjustments needed for freeze endurance remains unknown. 2. Recent studies using gene screening techniques are providing a different approach to the search for biochemical responses that support freezing survival by identifying genes and proteins that are up-regulated by freezing or thawing in freeze-tolerant amphibians and reptiles. 3. Screening of a cDNA library from wood frog liver revealed the freeze-induced up-regulation of genes coding for the alpha- and gamma-subunits of fibrinogen (a plasma clotting protein), the mitochondrial ADP/ATP translocase and a novel 10 kDa protein containing a nuclear exporting sequence. 4. Northern blotting revealed that these genes were differentially responsive to two of the component stresses of freezing (dehydration and anoxia), indicating that different genes are induced by signals radiating either from cell volume change or oxygen deprivation during freezing. 5. Freeze up-regulation of fibrinogen synthesis in liver and other organs appears to be a damage repair response that anticipates a need for enhanced plasma clotting capacity to deal with ice crystal damage to capillary beds. 6. Up-regulation of ADP/ATP translocase in frog liver is linked with ischaemia resistance and studies with freeze-tolerant turtles have shown that other genes encoding proteins involved in mitochondrial energetics (NADH-ubiquinone oxido-reductase subunit 5, cytochrome C oxidase subunit 1) are also up-regulated by both anoxia and freezing exposures. 7. These studies are making major advances in our understanding of freeze tolerance as a natural phenomenon and also highlight new key areas that can be targeted by applied interventions for the optimization of medical cryopreservation techniques for cells, tissues and organs.     

大鼠烫伤后早期肝脏线粒体RCR和ADP/O的变化

Sprague-Dawley(SD)大鼠(雄性)烫伤(相当于体表面积的20％,Ⅲ°)后30分钟组和烫伤后2小时组,其肝脏线粒体呼吸控制率(RCR)均比固定对照组和正常对照组高,差异显著;且烫伤后30分钟组肝脏线粒体氧化磷酸化效率(ADP/O)比固定对照组高,差异亦显著。此外从对32只雄性Wistar大鼠的测定结果中,也发现烫伤后30分钟组的肝脏线粒体RCR比固定对照组高,差异显著。SD大鼠烫伤后4小时组,其ADP/O比固定对照组低,差异十分显著;而RCR已接近正常。(大鼠肝脏线粒体活性测定时,均以琥珀酸盐为氧化底物)。说明大鼠烫伤后早期肝脏线粒体氧化磷酸化曾有偶联增强的时相。     

高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量

采用高效液相色谱法测定骨骼肌腺嘌呤核苷酸和辅酶Ⅰ含量。高压液相系 统包括：惠普公司ＺＯＢＡＸ－Ｃ１８反相柱５ｍｍ×１２ｃｍ，５９０ＵＶ检测器。流动相为２２０ｍｍｏｌ／Ｌ磷酸钾缓冲液，内含１０％甲醇，用四丁基氢氧化胺ＴＢＡＨ调ｐＨ至６５。等比洗脱，流速为０８ｍｌ／ｍｉｎ。检测波长为２５４ｎｍ。所有样品和标准品在进样前均用０４５μｍ孔径的虑膜过滤后进样５μｌ作色谱分析。结果表明：ＨＰＬＣ可以同时测定ＡＴＰ、ＡＤＰ、ＡＭＰ、ＮＡＤ＋、ＮＡＤＨ，分离效率高，操作简便，快速每个样品仅需１５分钟，而且，重现性好，定性可靠，定量准确。     

Regulation of human glutamate dehydrogenases: implications for glutamate, ammonia and energy metabolism in brain.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is probably involved in the metabolism of transmitter glutamate. The exact mechanisms that regulate glutamate fluxes through this pathway, however, have not been fully understood. In the human, GDH exists in heat-resistant and heat-labile isoforms, encoded by the GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) genes, respectively. These forms differ in their catalytic and allosteric properties. Kinetic studies showed that the K(m) value for glutamate for the nerve tissue GDH is within the range of glutamate levels in astrocytes (2.43 mM), whereas for the housekeeping enzyme, this value is significantly higher (7.64 mM; P < 0.01). The allosteric activators ADP (0.1-1.0 mM) and L-leucine (1.0-10.0 mM) induce a concentration-dependent enzyme stimulation that is proportionally greater for the nerve tissue-specific GDH (up to 1,600%) than for the housekeeping enzyme (up to 150%). When used together at lower concentrations, ADP (10-50 mM) and L-leucine (75-200 microM) act synergistically in stimulating GDH activity. GTP exerts a powerful inhibitory effect (IC(50) = 0.20 mM) on the housekeeping GDH; in contrast, the nerve tissue isoenzyme is resistant to GTP inhibition. Thus, although the housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Conditions associated with enhanced hydrolysis of ATP to ADP (e.g., intense glutamatergic transmission) are likely to activate nerve tissue-specific GDH leading to an increased glutamate flux through this pathway.     

NAD+-dependent mechanisms of disturbances of viability of brain cells during the acute period of hypoxic-ischemic perinatal injury

Using a model of perinatal hypoxic-ischemic injury of rat brain we have found the patterns of changes in the activity of ADP ribosyl cyclase in brain cells that determine the specific features of programmed cell death and maintenance of the intracellular NAD⁺ homeostasis.     

Decreased prostacyclin sensitivity of platelets in patients with Behçet''s syndrome

Abstract Patients with Behçet's syndrome have an increased risk of arterial and venous thrombosis, and abnormal platelet function has been implicated. Platelet function was studied in nine patients with Behçet's syndrome and in nine age- and sex-matched healthy volunteers. Platelet aggregation in response to ADP was measured, and the threshold concentration required to produce irreversible aggregation determined. Sensitivity of platelets to the inhibitory effect of prostacyclin was also determined. In addition, plasma levels of the platelet-specific proteins, β-thromboglobulin and platelet factor 4, and stimulated platelet thromboxane B₂ production, were measured. Platelets from patients with Behçet's syndrome showed normal aggregation in response to ADP, irrespective of disease activity. Platelet sensitivity to prostacyclin was, however, decreased compared with controls—with a mean prostacyclin ID₅₀ of 5·5 ± 1·3 ng ml^-1(mean ± SEM) and 1·9 ± 0·3 ng ml^-1, respectively (P < 0·01). This reduction in platelet sensitivity to prostacyclin was greatest in patients with the most active disease. These results suggest that Behçet's syndrome may be associated with altered platelet function, and this may have important consequences with regard to the increased risk of thrombosis associated with this condition.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

Drugs affecting coagulation

For more than half a century, heparin and vitamin K antagonists have defined anticoagulant therapy in both the short-term and long-term management of thrombotic diseases. However, the limitations of these traditional anticoagulants have prompted the development of new drugs. In the past 15 years new agents with improved safety profile and greater ease of use that target almost every step of the coagulation cascade have been developed. These include factor Xa inhibitors and direct thrombin inhibitors. The mechanism of action of these new anticoagulants and also the ‘older’ agents are reviewed in this article.