Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.     

Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells.

Na⁺ channels from lobster nerve membranes stored frozen in sucrose were incorporated into artificial liposomes. Crude soybean phospholipids or mixtures of purified phospholipids were suitable for reconstitution provided the latter included phosphatidylserine or another acidic phospholipid. The ²²Na flux into the reconstituted vesicles was increased (2 to 3-fold) by veratridine (0.25 – 1 mM) or grayanotoxin I (50 –150 μM) and the increment was abolished by 10 nM tetrodotoxin (K_i = 2 nM). The reconstituted vesicles were inactivated after incubation for 15 min at 40° and exposure to 20 μM dicyclohexylcardobiimide inhibited by 80% the response to the drugs.     

Identification of a novel splicing isoform of murine CGI-58

The comparative gene identification-58 (CGI-58) gene, mutations of which are linked to Chanarin-Dorfman syndrome, encodes a protein of the α/β hydrolase domain subfamily. We report here a new alternative splicing isoform of the murine CGI-58 gene, termed mCGI-58S. Sequence comparison indicates the lack of second and third exons in this cDNA variant. While the full-length protein displayed perilipin-dependent localization to lipid droplets, mCGI-58S showed a predominant cytoplasmic staining when expressed in cells. mCGI-58S was incapable of activating adipose triglyceride lipase but retained the capacity to acylate lysophosphatidic acid. Overexpression of mCGI-58S failed to promote lipid droplet turnover and loss of intracellular triacylglycerols. These results suggest that this splicing event may be involved in the regulation of lipid homeostasis.     

矫正型大动脉转位合并心内畸形的外科治疗

目的：为探讨矫正型大动脉转位的病理解剖特点及手术技术。方法：本组6例均为SLL型,手术包括：室间隔缺损修补4例、肺动脉瓣切开1例、静脉室肺动脉外通道1例、房室瓣替换1例。结果：全组手术死亡1例。主要手术并发症为低心排4例、完全性房室传导阻滞1例及残余左房室瓣关闭不全1例。结论：矫正型大动脉转位的病理解剖有一定的特殊性,应按不同的合并畸型选择不同术式,正确处理室间隔缺损、肺动脉流出道狭窄及左房室瓣关闭不全是外科手术的关键。     

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O₂ tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N₂, 5% CO₂) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD⁺ and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca²⁺ sensitivity to the myofilaments and diminished Ca²⁺-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca²⁺-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD^mut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP⁺ ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.     

XC1028 from Xanthomonas campestris adopts a PilZ domain‐like structure without a c‐di‐GMP switch

The crystal structure of XC1028 from Xanthomonas campestris has been determined to a resolution of 2.15 Å using the multiple anomalous dispersion approach. It bears significant sequence identity and similarity values of 64.10% and 70.09%, respectively, with PA2960, a protein indispensable for type IV pilus‐mediated twitching motility, after which the PilZ motif was first named. However, both XC1028 and PA2960 lack detectable c‐di‐GMP binding capability. Although XC1028 adopts a structure comprising a five‐stranded β‐barrel core similar to other canonical PilZ domains with robust c‐di‐GMP binding ability, considerable differences are observed in the N‐terminal motif; XC1028 assumes a compact five‐stranded β‐barrel without an extra long N‐terminal motif, whereas other canonical PilZ domains contain a long N‐terminal sequence embedded with an essential “c‐di‐GMP switch” motif. In addition, a β‐strand (β1) in the N‐terminal motif, running in exactly opposite polarity to that of XC1028, is found inserted into the parallel β3/β1′ strands, forming a completely antiparallel β4↓β3↑β1↓β1′↑ sheet in the canonical PilZ domains. Such dramatic structural differences at the N‐terminus may account for the diminished c‐di‐GMP binding capability of XC1028, and suggest that interactions with additional proteins are necessary to bind c‐di‐GMP for type IV fimbriae assembly. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Re-evaluating lipotoxic triggers in skeletal muscle: relating intramyocellular lipid metabolism to insulin sensitivity

Ectopic fat accumulation has been linked to lipotoxic events, including the development of insulin resistance in skeletal muscle. Indeed, intramyocellular lipid storage is strongly associated with the development of type 2 diabetes. Research during the last two decades has provided evidence for a role of lipid intermediates like diacylglycerol and ceramide in the induction of lipid-induced insulin resistance. However, recently novel data has been gathered that suggest that the relation between lipid intermediates and insulin resistance is less straightforward than has been previously suggested, and that there are several routes towards lipid-induced insulin resistance. For example, research in this field has shifted towards imbalances in lipid metabolism and lipid droplet dynamics. Next to imbalances in key lipogenic and lipolytic proteins, lipid droplet coat proteins appear to be essential for proper intramyocellular lipid storage, turnover and protection against lipid-induced insulin resistance.Here, we discuss the current knowledge on lipid-induced insulin resistance in skeletal muscle with a focus on the evidence from human studies. Furthermore, we discuss the available data that provides supporting mechanistic information.