The p53-regulated cyclin-dependent kinase inhibitor, p21 (cip1, waf1, sdi1), is not required for global genomic and transcription-coupled nucleotide excision repair of UV-induced DNA photoproducts.

The p53 tumor suppressor gene is a transcriptional activator involved in cell cycle regulation, apoptosis, and DNA repair. We have shown that p53 is required for efficient nucleotide excision repair of UV-induced DNA photoproducts from global genomic DNA but has no effect on transcription-coupled repair. In order to evaluate whether p53 influences repair indirectly through cell cycle arrest following DNA damage or plays a direct role, we examined repair in vivo in human cells genetically altered to disrupt or regulate the function of p53 and p21. Both primary human fibroblasts and HCT116 colon carcinoma cells wild type for p53 but in which the p21 gene was inactivated through targeted homologous recombination showed no decrease in global repair of UV photoproducts. Human bladder carcinoma cells mutant for p53 and containing a tetracycline-regulated p21 cDNA showed no significant enhancement of repair upon induction of p21 expression. All of the cell lines, including the mismatch repair-deficient, MLH1 mutant HCT116 cells, were proficient for transcription-coupled repair. Clonogenic survival of HCT116 cells following UV irradiation showed no dependence on p21. Therefore, our results indicate that p53-dependent nucleotide excision repair does not require the function of the p21 gene product and is independent of p53-regulated cell cycle checkpoints.

Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins.

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.

Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA. METHODS AND RESULTS: ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity. CONCLUSIONS: These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.

Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K).

Two naturally occurring mutants of human lecithin-cholesterol acyltransferase (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ovary cells, overproduced, and purified to homogeneity in order to study the structural and functional defects that lead to the LCAT deficiency phenotypes of these mutations. The mutants were expressed and secreted by transfected cells normally and had molecular weights and levels of glycosylation similar to wild type LCAT. The purified proteins (>98% purity) had almost indistinguishable structures and stabilities as determined by CD and fluorescence spectroscopy. Enzymatic activities and kinetic analysis of the pure enzyme forms showed that wild type LCAT and both mutants were reactive with the water-soluble substrate, p-nitrophenyl butyrate, indicating the presence of an intact core active site and catalytic triad. Both the T123I and N228K mutants had markedly depressed reactivity with reconstituted HDL (rHDL), but T123I retained activity with low density lipoprotein. To determine whether defective binding to rHDL was responsible for the low activity of both mutants with rHDL, the equilibrium binding constants were measured directly with isothermal titration calorimetry and surface plasmon resonance (SPR) methods. The results indicated that the affinities of the mutants for rHDL were only about 2-fold lower than the affinity of wild type LCAT (Kd = 2.3 x 10(-7) M). Together, the activity and equilibrium binding results suggest that the T123I mutant is defective in activation by apolipoprotein A-I, and the N228K mutant has impaired binding of lipid substrate to the active site. In addition, the kinetic binding rate constants determined by the SPR method indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.

Mutagenesis of highly conserved histidines in lecithincholesterol acyltransferase: identification of an essential histidine (His 377).

Lecithin-cholesterol acyltransferase (LCAT) is responsible for the formation of cholesterol esters in plasma and is implicated in the removal of excess cholesterol from peripheral tissues. It is generally accepted that the catalytic mechanism of LCAT is similar to that of serine proteases and lipases involving a Ser, a His, and an acidic amino acid residue. Ser181 in LCAT has been previously identified as a catalytic residue; however, the active site His and acidic residue have not yet been identified. In this study we have used a variety of approaches to identify the putative active site histidine. Alignments of LCAT sequences across various species indicate that the four histidines at positions 180, 263, 368, and 377 are conserved and could be involved in catalysis. Based on the observation that the members of the triad preserve the same orientation in the primary sequence of a large number of lipases, we eliminated His180 as a potential candidate. Mutational analysis along with functional assays show that, in contrast to the replacement of His263 and His368, the replacement of the His at position 377 with Gly, Ala, or Ser obliterates LCAT activity with interfacial and water-soluble substrates, thus indicating a role of His377 in catalysis.

Identification of a domain of lecithin-cholesterol acyltransferase that is involved in interfacial recognition.

Lecithin-cholesterol acyltransferase (LCAT) is an interfacial enzyme that acts on lipid substrates on the surface of high density lipoproteins (HDL). Based on observations with other interfacial lipases, we propose that LCAT contains a surface region of 25 amino acids linked by a disulfide bond (C50-C74) that is involved in the binding of LCAT to lipoproteins. Using LCAT cDNA, we have deleted most of this region (delta 53-71) and expressed the mutant enzyme (LCAT delta 53-71) in COS-1 cells. The deletion mutant is expressed and secreted at levels similar to wildtype LCAT, suggesting that the deleted region is located on the surface of the enzyme and is not required for folding. The enzymatic activity of the mutant was tested using two interfacial substrates, reconstituted HDL (rHDL) and low density lipoprotein (LDL), as well as a water soluble substrate, p-nitrophenyl butyrate (PNPB). There was no reaction with rHDL and LDL, but 30% of the activity with PNPB was retained. This suggests that the deleted region plays a role in interfacial binding, while the active site core is not disrupted. We thus conclude that this region (C50-C74) forms part of the interfacial binding domain of LCAT.

Analysis of human lecithin-cholesterol acyltransferase activity by carboxyl-terminal truncation.

Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (approximately 50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate, p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.

Theophylline in asthma management: current controversy.

Recent controversy regarding the use of theophylline has prompted this review of its role in the treatment of bronchospastic diseases. Salient points regarding theophylline's available preparations, pharmacology, metabolism, indications, side effects, and treatment of its toxicity are discussed.

Study of social, educational, environmental and cultural aspects of childhood asthma in clinic and private patients in the city of New York.

A group of asthmatics treated in a clinic setting were compared to a group treated by private allergists. Statistical differences were found regarding family income, racial distribution, family structure, educational level, reading habits, management of acute asthma attacks, housing, living space, school absences and hospital admissions.