Early referral and control of disease's flares prevent Orthopedic and Hand Surgery Indication (OHSI) in a dynamic cohort of Hispanic early rheumatoid arthritis patients.

BACKGROUND: Reconstructive joint surgery is an indicator of poor prognosis in rheumatoid arthritis (RA). Objectives of this study were to describe the incidence rate of orthopedic and hand surgery indication (OHSI) in an ongoing cohort of Hispanic early RA patients treated according to a T2T strategy and to investigate predictors. METHODS: Through February 2018, the cohort comprised 185 patients recruited from 2004 onwards, with variable follow-up, and rheumatic assessments at fixed intervals that included prospective determination of OHSI. Charts were reviewed by a single data abstractor. OHSI incidence rate was calculated. A case-control study nested within a cohort investigated the predictors; cases (OHSI patients) were paired with controls (1:4) according to age, sex and autoantibodies. A logistic regression model included baseline and cumulative (up to OHSI or equivalent) variables related to disease activity, treatment and to persistence with therapy. The IRB approved the study. RESULTS: Patients from the cohort were predominantly middle-aged (mean ± SD age: 38.5 ± 12.9 years) females (87.6%) with 5.4 ± 2.6 months of disease duration. The cohort contributed to 1538 patient-years of follow-up. Twelve patients received incidental OHSI at a follow-up of 85 ± 44.5 months. The OHSI incident global rate was 8/1000 patient-years. Longer symptom duration at cohort referral (OR: 1.313, 95%CI: 1.02-1.68, p = 0.032) and a higher number of flares/patient (OR: 1.608, 95%CI: 1.05-1.61, p = 0.015) predicted OHSI. OHSI patients had more severe flares than their counterparts, and the opposite figure was true for mild flares. CONCLUSION: Early referral for appropriate management and flare control may prevent OHSI in Hispanic recent-onset RA patients.

Mitochondrial cholesterol in health and disease.

Cholesterol is a critical component of biological membranes, which not only plays an essential role in determining membrane physical properties, but also in the regulation of multiple signaling pathways. Cells satisfy their need for cholesterol either by uptake from nutrients and lipoproteins or de novo synthesis from acetyl-CoA. The latter process occurs in the endoplasmic reticulum, where transcription factors that regulate the expression of enzymes involved in the de novo cholesterol synthesis reside. Cholesterol is distributed to different membranes most prominently to plasma membrane, where it participates in the physical organization of specific membrane domains. Mitochondria, however, are considered cholesterol-poor organelles, and obtain their cholesterol load by the action of specialized proteins involved in its delivery from extramitochondrial sources and trafficking within mitochondrial membranes. Although mitochondrial cholesterol fulfills vital physiological functions, such as the synthesis of bile acids in the liver or the formation of steroid hormones in specialized tissues, recent evidence indicates that the accumulation of cholesterol in mitochondria may be a key step in disease progression, including steatohepatitis, carcinogenesis or Alzheimer disease.

PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes.

A frameshifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induce cell death. Many proapoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic PB1-F2 (sPB1-F2) peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a transmembrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a severalfold increase in the rate of change of membrane conductance. sPB1-F2-treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite various experimental conditions, we did not detect the distinctive conductance levels typical of large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane-destabilizing profiles are seen with proapoptotic proteins (e.g., Bax) and the cytoplasmic helix of human immunodeficiency virus gp41, we suggest that the basis for sPB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis.

Membrane fusion: the process and its energy suppliers.

Membrane fusion constitutes a pivotal process in eukaryotic cell physiology. Both specialized proteins and membrane lipids play key roles in fusion. Here, our current understanding ofthe mechanism of membrane fusion is reviewed. The focus is on the relatively simple and well-understood proteinaceous fusion machinery of enveloped viruses and the physical properties of lipids that appear to be of great relevance for fusion progression. Recent observations suggest that viral fusion proteins use packed conformational energy and bilayer-destabilizing domains to (i) bring participating membranes into intimate contact, (ii) merge proximal lipid monolayers through highly curved stalk/hemifusion intermediates, and (iii) generate a lipid-containing fusion pore, thereby terminating the fusion process.

Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes.

During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. If Bcl-2 family proteins induce such release by increasing outer mitochondrial membrane permeability, then the pro-apoptotic, but not anti-apoptotic activity of these proteins should correlate with their permeabilization of membranes to cytochrome c. Here, we tested this hypothesis using pro-survival full-length Bcl-x(L) and pro-death Bcl-x(L) cleavage products (DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L)). Unlike Bcl-x(L), DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L) caused the release of cytochrome c from mitochondria in vivo and in vitro. Recombinant DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), as well as Bcl-x(L), cleaved in situ by caspase 3-possessed intrinsic pore-forming activity as demonstrated by their ability to efficiently permeabilize pure lipid vesicles. Furthermore, only DeltaN61Bcl-x(L) and DeltaN76Bcl-x(L), but not Bcl-x(L), formed pores large enough to release cytochrome c and to destabilize planar lipid bilayer membranes through reduction of pore line tension. Because Bcl-x(L) and its C-terminal cleavage products bound similarly to lipid membranes and formed oligomers of the same size, neither lipid affinity nor protein-protein interactions appear to be solely responsible for the increased membrane-perturbing activity elicited by Bcl-x(L) cleavage. Taken together, these data are consistent with the hypothesis that Bax-like proteins oligomerize to form lipid-containing pores in the outer mitochondrial membrane, thereby releasing intermembrane apoptogenic factors into the cytosol.

Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A.

Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations.

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore-the apoptotic pore-large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole.

Interbilayer lipid mixing induced by the human immunodeficiency virus type-1 fusion peptide on large unilamellar vesicles: the nature of the nonlamellar intermediates.

A peptide corresponding to the 23 N-terminal amino acid residues of the human immunodeficiency virus type-1 (HIV-1) gp41 has the capacity to induce intervesicular lipid mixing in large unilamellar liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol (CHOL) (molar ratio, 1:1:1). Cryo-transmission electron microscopy (cryo-TEM) of diluted vesicles to which peptides has been externally added reveals a morphology that is compatible with the formation of nonlamellar lipidic aggregates during the time-course of lipid mixing. 31P-nuclear magnetic resonance and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMADPH) steady-state anisotropy data at equilibrium indicate that the peptide is able to modulate the lipid polymorphism in pelletted membranes by: (i) promoting the thermotropic formation of inverted phases; and (ii) driving the lamellar-to-nonlamellar transition towards the formation of isotropic phases. Therefore, our combined morphological and spectroscopic data reveal the existence of a direct correlation between the ability of the externally added peptide to induce lipid-mixing in dilute liposome samples and its capacity to modulate lipid polymorphism in stacked bilayers.

Effect of single chain lipids on phospholipase C-promoted vesicle fusion. A test for the stalk hypothesis of membrane fusion.

The effect of low proportions (up to 5 mol %) of single-chain lipids on phospholipase C-promoted fusion of large unilamellar vesicles has been investigated with the aim of testing the so-called stalk model of membrane fusion. This model is known in two main versions, the one originally published by Kozlov and Markin [Kozlov, M. M. and Markin, V. S. (1983) Biofizika 28, 255-261] and what is known as the "modified stalk model" [Siegel, D. P. (1993) Biophys. J. 65, 2124-2140], that differ in a number of predictions. In the view of the latter author, hydrocarbons or other nonpolar lipids should help fusion by decreasing the interstitial energy of the stalk connecting the two apposed bilayers. We show that small amounts of hexadecane or squalene increase significantly the fusion rates in our system. Changes in monolayer curvature are the object of different predictions by the original and modified stalk theories. According to the original form, fusion would be promoted by lipids inducing a negative curvature in the closest (cis) monolayers of the fusing membranes and inhibited by the same lipids in the trans monolayers; the opposite would happen with lipids inducing a positive curvature. The modified stalk model predicts that fusion is helped by increasing the negative curvature of both monolayers. In our system, symmetrically distributed arachidonic acid, which increases the negative curvature, enhances lipid and content mixing, and the opposite is found with symmetrically distributed lysophosphatidylcholine or palmitoylcarnitine, which facilitate a positive monolayer curvature. In addition, fluorescence polarization and 31P NMR studies of the lamellar-to-isotropic (Q224 cubic) thermotropic transition of a lipid mixture corresponding to our liposomal composition reveal that all lipids that facilitate fusion decrease the transition temperature, while fusion inhibitors increase the transition temperature. Moreover, fusion (content mixing) rates show a maximum at the lamellar-to-isotropic transition temperature. These observations support the involvement of inverted lipid structures, as occurring in the inverted cubic phases, in membrane fusion. All these data are in full agreement with the stalk model of membrane fusion, particularly in its modified version.

Interfacial enzyme activation, non-lamellar phase formation and membrane fusion. Is there a conducting thread?

Previous studies from this laboratory have shown that the enzymic generation of diacylglycerol in bilayers by phospholipase C may lead to membrane fusion through the formation of transient non-lamellar lipidic intermediates. The present paper intends to explore the correlations existing among the three main processes involved, namely (a) the induction (or inhibition) of lamellar-to-non-lamellar phase transitions in lipid mixtures through the addition of small (< 5 mol%) proportions of other lipids, (b) the promotion, by the latter lipids, of fusion in otherwise stable phospholipid vesicles (large unilamellar liposomes) under conditions leading to inverted hexagonal/inverted cubic phase formation in bulk lipid systems, and (c) the modulation, by the same small proportions of lipids, of phospholipase C hydrolysis of phosphatidylcholine in liposome bilayers. It is concluded that phospholipase C may give rise to non-lamellar lipidic structures that in turn permit liposomal fusion to occur, but neither enzyme activity is directly modulated by non-lamellar phase formation, nor will whatever kind of enzyme-induced non-lamellar structure give rise to fusion. Moreover, only under certain kinetic conditions will the enzyme give rise to the organization of non-lamellar structures that are conducive to the fusion event.

Poly(ethylene glycol)-lipid conjugates inhibit phospholipase C-induced lipid hydrolysis, liposome aggregation and fusion through independent mechanisms.

Poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE) conjugates have been introduced in liposomal compositions. The resulting large unilamellar vesicles were subjected to the action of phospholipase C. Enzyme-promoted vesicle aggregation and fusion were assayed in liposomes containing various proportions of PEG-PE. At PEG-PE concentrations above 1 mol% the rate of phospholipid hydrolysis decreases, perhaps because the PEG moiety hinders the enzyme from reaching the membrane surface. At concentrations above 0.1 mol% vesicle aggregation occurs at a slower rate, presumably because of the repulsive barrier properties or surface-grafted PEG. Lipid mixing decreases in parallel with vesicle aggregation. Finally, liposomal fusion rates measured as mixing of vesicle aqueous contents are decreased at or even below 0.1 mol%. The latter inhibition is due, apart from the reduced rates of lipid hydrolysis, vesicle aggregation and lipid mixing, to a PEG-PE-based stabilization of the lipid bilayer structure. Thus the observed low rates of contents mixing arise from three combined and independent inhibitory effects of PEG-PE.

Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion.

Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.

Origin of the lag period in the phospholipase C cleavage of phospholipids in membranes. Concomitant vesicle aggregation and enzyme activation.

When phospholipase C is added to a suspension of large unilamellar vesicles of egg phosphatidylcholine, maximal rates of hydrolysis occur only after a latency period. No lag period is seen when the substrate is in the form of small (sonicated) vesicles, or of short-chain phosphatidylcholine monomers. For a given vesicle concentration, the lag time may vary as a function of Ca2+, enzyme concentration, or temperature, but activation occurs at a fixed molar fraction of diacylglycerol produced. Lag times decrease gradually with vesicle size, and also with the amount of diacylglycerol present in the bilayers when it is mixed with phospholipid prior to enzyme addition. Parallel recordings of enzyme activity and suspension turbidity reveal that in all cases the latency period ends concomitantly with the start of a process of vesicle aggregation. Both the lag time and the amount of diacylglycerol formed before activation decrease with vesicle concentration, suggesting that enzyme activation is somehow related to vesicle aggregation. The latency period of phospholipase C may be explained in terms of a hypothesis according to which (a) full enzyme activity requires the presence of membrane surface irregularities or defects, (b) the diacylglycerol generated in the lag phase produces some kind of phase separation, with the formation of diacylglycerol-rich "patches" or domains, (c) vesicles aggregate through contacts between those patches, and (d) aggregation causes (and/or increases, and/or stabilizes) the surface inhomogeneities that allow fast enzyme activity. These data and suggestions may be relevant to the process of model membrane fusion promoted by phospholipase C.

Different effects of enzyme-generated ceramides and diacylglycerols in phospholipid membrane fusion and leakage.

When large unilamellar vesicles consisting of sphingomyelin:phosphatidylethanolamine:cholesterol (2:1:1 molar ratio) are treated with sphingomyelinase, production of ceramides in the bilayer is accompanied by leakage of vesicle aqueous contents and by vesicle aggregation in the absence of lipid mixing or vesicle fusion. This is in contrast to the situation of phosphatidylcholine:phosphatidylethanolamine:cholesterol (2:1:1 molar ratio) liposomes when treated with phospholipase C. In that case, in situ generation of diacylglycerol leads to vesicle aggregation followed by vesicle fusion in the absence of leakage (Nieva, J. L., Goñi, F. M., and Alonso, A. (1989) Biochemistry 28, 7364-7367). Moreover, when ceramides (5-10 mol %) are included in the formulation of the phosphatidylcholine-containing vesicles, they reduce the lag time of phospholipase C-induced fusion, although they are less active than diacylglycerols in this respect. 31P NMR studies of aqueous lipid dispersions show that diacylglycerols as well as ceramides induce a thermotropic lamellar to non-lamellar phase transition in both phospholipid:cholesterol mixtures under study although sphingomyelin-containing bilayers are more stable than those containing phosphatidylcholine, and ceramide is less active than diacylglycerol in promoting non-lamellar phase formation. These observations are relevant to both the physiological role of ceramides and the current views on the mechanism of membrane fusion.

Dual inhibitory effect of gangliosides on phospholipase C-promoted fusion of lipidic vesicles.

The effect of a variety of gangliosides has been tested on the phospholipase C-induced fusion of large unilamellar vesicles. Bilayer composition was phosphatidylcholine:phosphatidylethanolamine: cholesterol (2:1:1 mole ratio) plus the appropriate amounts of glycosphingolipids. Enzyme phosphohydrolase activity, vesicle aggregation, mixing of bilayer lipids and mixing of liposomal aqueous contents were separately assayed. Small amounts ( < 1 mol %) of gangliosides in the lipid bilayer produce a significant inhibition of the above processes. The inhibitory effect of gangliosides increases with the size of the oligosaccharide chain in the polar head group. Inhibition depends in a nonlinear manner on the ganglioside proportion, and is complete at approximately 5 mol %. Inhibition is not due to ganglioside-dependent changes in vesicle curvature or size. Ganglioside inhibition of vesicle fusion is due to two different effects: inhibition of phospholipase C activity and stabilization of the lipid lamellar phase. Enzyme inhibition leads to a parallel decrease of vesicle aggregation and lipid mixing rates. Mixing of aqueous contents, though, is depressed beyond the enzyme inhibition levels. This is explained in terms of the fusion pore requiring a local destabilization of the lipid bilayer, the lamellar structure being stabilized by gangliosides. 31P-NMR and DSC experiments confirm the inhibitory effect of gangliosides in various lamellar-to-nonlamellar transitions.

Diacylglycerol and the promotion of lamellar-hexagonal and lamellar-isotropic phase transitions in lipids: implications for membrane fusion.

Changes in steady-state fluorescence anisotropy of 1 -(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene TMA-DPH) are applied to the detection of lamellar-hexagonal transitions in egg phosphatidylethanolamine. Even low (2 mole%) proportions of diacylglycerol decrease the hexagonal transition temperature considerably, as confirmed by differential scanning calorimetry. Diacylglycerol is also found to promote a lamellar to "isotropic" (Q(224) cubic) transition in mixtures of phosphatidylcholine: phosphatidylethanolamine:cholesterol. This nonreversible transition is also observed by (31)P nuclear magnetic resonance and detected as a large increase in TMA-DPH steady-state anisotropy. The same technique reveals as well that lysophosphatidylcholine counteracts the effect of diacylglycerol and stabilizes the lamellar phase in both transitions. Diacylglycerol and lysophosphatidylcholine are known to respectively promote and inhibit membrane fusion in a variety of systems. These data are interpreted in support of the hypothesis of a highly bent structural fusion intermediate ("stalk"). They also show the interest of lipid-phase studies in predicting and rationalizing membrane fusion mechanisms.

Topological properties of two cubic phases of a phospholipid:cholesterol:diacylglycerol aqueous system and their possible implications in the phospholipase C-induced liposome fusion.

Water dispersions of phospholipid:cholesterol:diacylglycerol may, under certain conditions, originate either the lipid- and water-permeable Q224 cubic phase, or the lipid-permeable but water-impermeable Q227 cubic phase. These results are discussed within the framework of the phospholipase C-induced fusion of liposomes [Nieva et al. (1993) Biochemistry 32, 1054]. It is suggested that the cubic phases Q224 and Q227 represent two classes of lipid organization, one promoting, the other hindering the mixing of aqueous contents that is characteristic of membrane fusion. In this context, inverted micelles appear to be the end point of the fusion process, rather than fusion intermediates.