Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning

Although it is well documented that neutrophils are critical for the delayed phase of hepatic ischemia-reperfusion injury, there is no direct evidence for a specific neutrophil-derived oxidant stress in vivo. Therefore, we used a model of 60 min of partial hepatic ischemia and 0-24 h of reperfusion to investigate neutrophil accumulation and to analyze biomarkers for a general oxidant stress [glutathione disulfide (GSSG) and malondialdehyde (MDA)] and for a neutrophil-specific oxidant stress [hypochlorite (HOCl)-modified epitopes] in rats. Plasma alanine transaminase activities and histology showed progressively increasing liver injury during reperfusion, when hepatic GSSG and soluble MDA levels were elevated. At that time, few neutrophils were present in sinusoids. However, the number of hepatocytes positively stained for HOCl-modified epitopes increased from 6 to 24 h of reperfusion, which correlated with the bulk of hepatic neutrophil accumulation and extravasation into the parenchyma. Consistent with a higher oxidant stress at later times, hepatic GSSG and protein-bound MDA levels further increased. Treatment with the NADPH oxidase inhibitor diphenyleneiodonium chloride attenuated postischemic oxidant stress (GSSG, protein-bound MDA, and hepatocytes positively stained for HOCl-modified epitopes) and liver injury at 24 h of reperfusion. Ischemic preconditioning suppressed all oxidant stress biomarkers, liver injury, and extravasation of neutrophils. In conclusion, extravasated neutrophils generate HOCl, which diffuses into hepatocytes and causes oxidative modifications of intracellular proteins during the neutrophil-mediated reperfusion injury phase. Ischemic preconditioning is an effective intervention for reduction of the overall inflammatory response and, in particular, for limitation of the cytotoxic activity of neutrophils during the later reperfusion period.     

Experience with a thin-layer,liquid-based cervical cytologic screening method

OBJECTIVE: To assess the utility of a thin-layer cytology system for cervicovaginal screening in clinical practice. STUDY DESIGN: Twenty-five hundred cervicovaginal split samples were processed with the traditional direct smearing method and with the ThinPrep Pap Test (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) method and evaluated according to the Bethesda system, focusing mainly on the cytomorphologic features. RESULTS: The ThinPrep Pap Test yielded improved specimen adequacy and an increased detection rate of squamous abnormalities, which resulted in a decrease in the ASCUS/SIL ratio. Moreover, the thin-layer system provided adequate material for concomitant HPV testing, mainly in the LSIL and the ASCUS favor SIL cases, with satisfactory results, as well as for cell block preparations in a few selected cases that presented diagnostic difficulties. Furthermore, the morphologic features of the LSIL cases were virtually identical on both preparations, while in the HSIL cases, distinct features were noted on ThinPrep. CONCLUSION: The ThinPrep Pap Test is significantly more effective than the conventional smear in clinical practice. However, training and experience are necessary to take full advantage of this promising new technology.     

Plasma phospholipid transfer protein-mediated reactions are impaired by hypochlorite-modification of high density lipoprotein

The two main functions of phospholipid transfer protein (PLTP) are the transfer of phospholipids between plasma lipoproteins and the conversion of high density lipoprotein (HDL), where prebeta-HDL particles are generated. HDL is considered an anti-atherogenic lipoprotein due to its function in the reverse cholesterol transport, where prebeta-HDL accepts cellular membrane cholesterol from peripheral tissues. However, the anti-atherogenic properties of native HDL may be abolished by oxidation/modification. Hypochlorous acid/hypochlorite (HOCl/OCl-)-a potent oxidant generated in vivo only by the myeloperoxidase-H2O2-chloride system of activated phagocytes-alters the physiological properties of HDL by generating a pro-atherogenic lipoprotein particle. Therefore, we have studied the effect of HOCl on the function of HDL subclass 3 (HDL3) and triglyceride-enriched HDL3 (TG-HDL3) in PLTP-mediated processes in vitro. Modification of HDL3 and TG-HDL3 with increasing HOCl concentrations (oxidant:lipoprotein molar ratio between 25:1 and 200:1) decreased the capacity of the corresponding lipoprotein particles to accept phospholipids. Although binding of PLTP to unmodified and HOCl-modified lipoprotein particles was similar, the degree of PLTP-mediated HDL conversion was decreased upon HOCl oxidation. PLTP released apolipoprotein A-I (apoA-I) from HOCl-modified HDL3, but the particles formed displayed no prebeta-mobility. Based on these findings, we conclude that the substrate properties of HOCl-modified HDL3 and TG-HDL3 in PLTP-mediated processes are impaired, which indicates that the anti-atherogenic properties of HDL are impaired.     

Uptake and transport of high-density lipoprotein (HDL) and HDL-associated alpha-tocopherol by an in vitro blood-brain barrier model

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high-density lipoproteins (HDLs) and HDL-associated alpha-tocopherol (alpha TocH) across an in vitro model of the blood-brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL-associated alpha TocH was taken up in 10-fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR-BI). SR-BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl-beta-cyclodextrin (CDX) resulted in (mis)sorting of SR-BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR-BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL-associated [(14)C]alpha TocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin-1, the major caveolae-associated protein. During mass transfer studies using alpha TocH-enriched HDL, approximately 50% of cellular alpha TocH was recovered with the bulk of cellular caveolin-1 and SR-BI. Efflux experiments revealed that a substantial amount of cell-associated [(14)C]alpha TocH could be mobilized into the culture medium. In addition, apical-to-basolateral transport of HDL holoparticles and HDL-associated alpha TocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A-I and alpha TocH originates from plasma HDL transcytosed across the BBB and that caveolae-located SR-BI facilitates selective uptake of HDL-associated alpha TocH at the BBB.     

Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.     

Mule deer (Odocoileus hemionus) respond to yellow‐bellied marmot (Marmota flaviventris) alarm calls

Individuals may obtain valuable information about the presence of predators by listening to heterospecific alarm signals. Most playback studies have demonstrated that similarly sized and taxonomically related species may respond to the calls of each other, but less work has been carried out to define these factors influence responsiveness to alarm signals. In theory, individuals should respond to calls from any species that provide information about the presence of important predators, regardless of body size or taxonomic relationship. However, size is often associated with vulnerability. Coyotes (Canis latrans) in the Rocky Mountains prey upon both mule deer (Odocoileus hemionus) and yellow‐bellied marmots (Marmota flaviventris), which differ considerably in size, alarm vocalizations, and antipredator behavior. We conducted a playback experiment to see whether deer discriminated between marmot alarm calls and the non‐alarm song of a common sympatric bird. We found that deer increased vigilance significantly more after hearing broadcast marmot alarm calls compared with the bird song. Interestingly, deer that were studied within 0.5 km of homes showed significantly greater discrimination than those studied farther from humans. Our results suggest relative size differences do not prevent interspecific communication and that common predators should generally drive the evolution of the ability to learn to respond to meaningful risk cues. As long as two species share a predator, it should benefit the other to respond to its alarm calls.     

Distinct HDL subclasses present similar intrinsic susceptibility to oxidation by HOCl

The heme protein myeloperoxidase (MPO) functions as a catalyst for lipoprotein oxidation. Hypochlorous acid (HOCl), a potent two-electron oxidant formed by the MPO-H₂O₂-chloride system of activated phagocytes, modifies antiatherogenic high-density lipoprotein (HDL). The structural heterogeneity and oxidative susceptibility of HDL particle subfractions were probed with HOCl. All distinct five HDL subfraction were modified by HOCl as demonstrated by the consumption of tryptophan residues and free amino groups, cross-linking of apolipoprotein AI, formation of HOCl-modified epitopes, increased electrophoretic mobility and altered content of unsaturated fatty acids in HDL subclasses. Small, dense HDL3 were less susceptible to oxidative modification than large, light HDL2 on a total mass basis at a fixed HOCl:HDL mass ratio of 1:32, but in contrast not on a particle number basis at a fixed HOCl:HDL molar ratio of 97:1. We conclude that structural and physicochemical differences between HDL subclasses do not influence their intrinsic susceptibility to oxidative attack by HOCl.