The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.     

Cellular remodeling in heart failure disrupts K(ATP) channel-dependent stress tolerance

ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K(ATP) channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.

     

Glycoprotein IIb/IIIa receptor antagonists in acute myocardial infarction and primary coronary stenting: Not always effective

Glycoprotein IIb/IIIa antagonists have been shown to be effective in reducing thrombotic complications prior to high-risk coronary interventions. Some studies have reported improved coronary flow after abciximab in slow or no-reflow phenomenon. We report a case in which abciximab did not clear the thrombotic occlusion or restore artery flow. Further studies are needed into the refractory no-flow phenomenon.     

Ligand-insensitive State of Cardiac ATP-sensitive K+ Channels : Basis for Channel Opening

The mechanism by which ATP-sensitive K⁺ (K_ATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac K_ATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. “Rundown” of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the K_ATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac K_ATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for K_ATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

ESR Studies of a Series of Phthalocyanines. Mechanism of Phototoxicity. Comparative Quantitation of O2-using ESR Spin-Trapping and Cytochrome c Reduction Techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperi-done (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexyl-siloxy) silicon phthalocyanine ([(nhex)₃SiO]₂SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl₁₆), the hexadecachloro aluminum phthalocyanine (AlPcCl₁₆), the hexadecachloro aluminum phthalocyanine sulfate (HSO₄A1PcCl₁₆), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlori-nated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexyl-siloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.     

Discrete consumers,small scale resource heterogeneity,and population stability

We present a consumer-resource model in which individual consumers subsist on a continuum of resource distributed over a very large number of small “bite-sized” patches, each patch being sufficiently small that all its resource is eaten whenever a consumer visits. This form of consumer–resource interaction forces a heterogeneous distribution of resource among the patches, and may dampen out the large amplitude, consumer-resource cycles that are predicted by traditional models of well-mixed, spatially homogeneous systems. The resource equilibrium does not increase with enrichment, a prediction that distinguishes this model from models that invoke direct or indirect consumer density dependence as a stabilizing mechanism.     

Rib cage muscle interaction in airway pressure generation

We have previously demonstrated in dogs that the change inairway opening pressure (Pao) produced by isolated maximumactivation of the parasternal intercostal or triangularis sterni musclein a single interspace, the sternomastoids, and the scalenes isproportional to the product of muscle mass and the fractional change inmuscle length per unit volume increase of the relaxed chest wall. In the present study, we have assessed the interactions between these muscles by comparing the Pao obtained during simultaneous activation of a pair of muscles (measured Pao) to the sum of the Pao values obtained during their separate activation (predicted Pao). Measured and predicted Pao values were compared for the following pairs ofmuscles: the parasternal intercostals in two interspaces, the parasternal intercostals in one interspace and either thesternomastoids or the scalenes, two segments of the triangularissterni, and the interosseous intercostals in two contiguousinterspaces. For all these pairs, the measured Pao was within~10% of the predicted value. We therefore conclude that1) the pressure changes generated bythe rib cage muscles are essentially additive; and2) measurements of the mass of aparticular muscle and of its fractional change in length during passiveinflation can be used to estimate the potential pressure-generatingability of the muscle during coordinated activity as well as duringisolated activation.