Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1–189 and 190–243) and mouse apoA-I (residues 1–186 and 187–240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.     

High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus

Introduction

Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.

Methods

Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of ³⁵S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, β2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.

Results

Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.

Conclusions

Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.     

Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens

Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.     

Chlorin p6 as a fluorescent probe for the investigation of surfactant-cyclodextrin interactions.

Cyclodextrins (CD) are often proposed as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity/lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p6, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between alpha-CD and beta-CD with the neutral surfactants Triton X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). The fluorophore is found to be a sensitive one for the study of the interaction of alpha, beta and gamma-CD with the surfactants TX 100 and CTAB. It is found that contrary to earlier reports, a complex between alpha-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p6, which does not bind to the CDs, but not with a fluorophore, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and alpha-CD as compared to that between TNS and CD.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells

Introduction  

The aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.