The acute effect of smoking in healthy and asthmatic smokers.

BACKGROUND: Acute exposure to cigarette smoke is related to airway and systemic inflammation and oxidative stress. Little is known about the acute effect of cigarette smoking in smoking asthmatics. The aim of this study was to evaluate the acute effect of smoking in airway and systemic inflammation and oxidative stress in normal smokers and patients with properly treated well-controlled persistent asthma. MATERIALS AND METHODS: Ten normal smokers and 10 smokers with moderate persistent asthma controlled with LABA and ICS were recruited. Subjects refrained from smoking for at least 12 h prior to their inclusion. We compared the effects of smoking of two cigarettes on airway obstruction, airway inflammation and oxidative stress [by measuring fraction of exhaled nitric oxide (FeNO), plus pH and 8-isoprostane in exhaled breath condensate (EBC)] before and 30, 90 and 180 min after smoking. Furthermore, we evaluated systemic oxidative stress, C-reactive protein (CRP) and serum amyloid A (SAA) and urine leukotriene E(4) (LTE(4)) before and 180 min after smoking. RESULTS: No differences were observed in EBC pH and 8-isoprostane, FeNO and systemic oxidative stress between the groups at baseline. In asthmatics, EBC pH decreased 30 min and EBC 8-isoprostane increased 90 min after smoking (P = 0.039 and P = 0.029 respectively), which was not evident in smoking controls. Serum oxidative stress increased only in asthmatic smokers at 180 min (P = 0.001). No differences were observed in SAA, CRP and urine LTE(4) levels before and after smoking. CONCLUSION: Acute smoking has more deleterious effects in well-controlled properly treated asthmatic smokers compared with matched normal smokers.

Side population cells isolated from different tissues share transcriptome signatures and express tissue-specific markers.

Side Population (SP) cells, isolated from murine adult bone marrow (BM) based on the exclusion of the DNA dye Hoechst 33342, exhibit potent hematopoietic stem cell (HSC) activity when compared to Main Population (MP) cells. Furthermore, SP cells derived from murine skeletal muscle exhibit both hematopoietic and myogenic potential in vivo. The multipotential capacity of SP cells isolated from variable tissues is supported by an increasing number of studies. To investigate whether the SP phenotype is associated with a unique transcriptional profile, we characterized gene expression of SP cells isolated from two biologically distinct tissues, bone marrow and muscle. Comparison of SP cells with differentiated MP cells within a tissue revealed that SP cells are in an active transcriptional and translational status and underexpress genes reflecting tissue-specific functions. Direct comparison of gene expression of SP cells isolated from different tissues identified genes common to SP cells as well as genes specific to SP cells within a particular tissue and further define a muscle and bone marrow environment. This study reports gene expression of muscle SP cells, common features and differences between SP cells isolated from muscle and bone marrow, and further identifies common signaling pathways that might regulate SP cell functions.

In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.

Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.

To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.

Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association and APOA-I mutations on receptor binding.

The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with (125)I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K(d) value for spherical HDL (density = 1.1-1.13 g/ml) was approximately 16 microgram of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (approximately 0.4-5 microgram of protein/ml). We also observed reduced affinity and/or competition for spherical (125)I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical (125)I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH(2) and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.

Structure and function of apolipoprotein A-I and high-density lipoprotein.

Structural biology and molecular modeling have provided intriguing insights into the atomic details of the lipid-associated structure of the major protein component of HDL, apo A-I. For the first time, an atomic resolution map is available for future studies of the molecular interactions of HDL in such biological processes as ABC1-regulated HDL assembly, LCAT activation, receptor binding, reverse lipid transport and HDL heterogeneity. Within the context of this paradigm, the current review summarizes the state of HDL research.