Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.     

Analysis of digital patterns in the Basque valley of Deba: multivariate comparison with other populations

Eighty-six females and 79 males from the Basque valley of Deba were analyzed with respect to their digital patterns using correspondence analysis. We found that there was a significant sexual difference for only one type of pattern, tented arch. Also, this population was compared with other Basque valley populations and with other Spanish populations. We found great variability among Basque subpopulations, despite their supposed common origin. Dermatoglyphic distances for some traits can be interpreted as genetic distances because there is high heritability of these traits. The results of the comparison between the Deba valley population and other Spanish populations showed that the Deba population is markedly distant from the other populations.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.