Prevalence of oral soft tissue lesions in HIV-infected minority children treated with highly active antiretroviral therapies.

PURPOSE: This project studied the prevalence of oral soft tissue disease in HIV-infected children treated with highly active antiretroviral therapy (HAART). METHODS: Thirty-eight HIV-infected children participated in the study. Twenty-three of these patients were treated with HAART while 14 received exclusively reverse transcriptase inhibitors (RTI) and served as controls. The children were examined three times at approximately one-month intervals while their health history and laboratory data were abstracted from medical charts. Analyses were performed to determine differences in lesion prevalence between treatment groups as well as between lesion and no lesion groups with regard to immune differences. RESULTS: Thirty patients (79%) had oral lesions detected in at least one visit. There were no differences in specific lesion prevalence between HAART compared with RTI-treated children. However, a trend for more oral candidiasis in the latter group was observed. Subjects with oral soft tissue lesions had lower CD4 counts (P = 0.04) and percentage (P = 0.01) but similar viral loads when compared to patients without oral soft tissue disease. CONCLUSIONS: HAART does not appear to significantly affect oral soft tissue disease prevalence in HIV-infected children. Presence of lesions was associated with decreased immunity and may signal advancing disease.

Regulation of ornithine decarboxylase mRNA levels in human breast cancer cells: pattern of expression and involvement of core enhancer promoter element.

Ornithine decarboxylase (ODC) expression is increased by growth factors and is obligatory for progression through the cell cycle in a wide variety of cell types. In this study, a variant human ODC cDNA was identified, sequenced, and used to probe mRNA levels in human breast tumor cell lines and xenografts. ODC mRNA was elevated about 3-fold in estrogen receptor-negative (ER-) tumors (MDA-MB-231) when compared with ER-positive (ER+) tumors (MCF-7), as assessed by quantitative autoradiographic analysis of in situ hybridization experiments. The pattern of ODC mRNA in MDA-MB-231 (ER-) xenografts was polarized to the extreme periphery of the tumor, whereas the distribution of ODC mRNA was more evenly distributed in MCF-7 (ER+) xenografts. This correlates with hematoxylin and eosin staining patterns, suggesting that ER+ and ER- xenografts have a differential dependence on host vasculature for growth factor supply. ODC mRNA was elevated 5-fold in MDA-MB-231 cells versus MCF-7 cells when analyzed in cell culture. These relative mRNA levels correlate with increased levels of "core" enhancer binding nuclear proteins in MDA-MB-231 cells over that detected in MCF-7 cells.

An alternative mechanism for the inhibition of cholesterol biosynthesis in HepG2 cells by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815).

Compounds that block hepatic cholesterol biosynthesis and secretion may be useful hypocholesterolemic agents. N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815) has been shown to block cholesterol biosynthesis in 3T3 fibroblasts and it causes cellular accumulation of squalene 2,3-epoxide and squalene 2,3:23,24-diepoxide (squalene epoxides), which suggests that it inhibits 2,3-oxidosqualene cyclase. The purpose of the present report was to determine whether MDL 28,815 acts only at the level of 2,3-oxidosqualene cyclase or whether other enzymes in the cholesterol biosynthetic pathway are affected. HepG2 cells, grown in lipoprotein-deficient serum, were incubated with MDL 28,815 and 14C-acetate to radiolabel cholesterol and the intermediates in the cholesterol biosynthetic pathway. Blockade of cholesterol biosynthesis by MDL 28,815 in these cells was associated with the accumulation of two metabolites, one of which was 5 alpha-cholest-8-en-3 beta-ol. The other metabolite was identified by a combination of ultraviolet spectrometry, gas chromatography, mass spectroscopy and analytical high-performance liquid chromatography as 5 alpha-cholest-8,14-dien-3 beta-ol. Maximal blockade of cholesterol biosynthesis was associated with the accumulation of these two metabolites and, in particular, 5 alpha-cholest-8,14-dien-3 beta-ol, rather than with squalene epoxides. These results suggest that MDL 28,815 blocks cholesterol biosynthesis primarily by the inhibition of sterol-delta 14-reductase, and possibly sterol-delta 8-ene isomerase, rather than 2,3-oxidosqualene cyclase.

Similarities and differences between the interleukin-1 induction and action pathways in human macrophages.

Cyclic AMP, protein kinase A and NF kappa B have been implicated as second messengers in the interleukin-1 (IL-1) action pathway. Since IL-1 induces more IL-1 release, the IL-1 action pathway may share some common second messengers with the IL-1 induction pathway. Therefore, we investigated whether cyclic AMP, protein kinase A and NF kappa B are involved in the induction of IL-1 beta release by human peripheral blood monocyte-derived macrophages (HPBM) stimulated with a specific IL-1 beta inducer, 9-hydroxyoctadecadienoic acid (9-HODE). With regard to cyclic AMP, it peaked 30 min after 9-HODE stimulation. A role for cyclic AMP in IL-1 beta induction was suggested since forskolin was sufficient to induce IL-1 beta release from HPBM. 9-HODE stimulation of HPBM also activated an early peak of protein kinase A activity. A requirement of protein kinase A in IL-1 beta induction was suggested since 9-HODE-induced IL-1 beta release was inhibited with a selective protein kinase A inhibitor, Rp-isomer (IC50:5 microM). Lastly, to examine the role of NF kappa B, incubation of HPBM with a double-stranded oligodeoxyribonucleotide (ds-oligo) bearing the NF kappa B consensus sequence produced a dose-dependent enhancement of 9-HODE-induced IL-1 beta release, whereas a ds-oligo containing an unrelated Oct-1 motif had no effect. These results suggest that NF kappa B plays a negative role in the IL-1 beta induction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture.

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualene N-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol synthesis in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [3H]-squalene conversion to [3H]squalene epoxide in microsomes (IC50 = 5.0 microM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualene N-methylcyclopropylamine inhibited [3H]squalene conversion to [3H]lanosterol (IC50 = 12.0 microM) and caused [3H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [14C]acetate in HepG2 cells was inhibited by both derivatives (IC50 = 1.0 microM for trisnorsqualene cyclopropylamine; IC50 = 0.5 microM for trisnorsqualene N-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [14C]squalene, while cells incubated with trisnorsqualene N-methylcyclopropylamine accumulated [14C]squalene epoxide and [14C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.

Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3-methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2.

The level of hepatic triglyceride lipase (H-TGL) synthesis and secretion was examined in response to changes in cholesterol biosynthesis in the human hepatoma cell line HepG2. Cells were first fed a lipoprotein-deficient serum-supplemented medium to eliminate exogenous cholesterol. Mevinolin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, was then added at a concentration (37 microM) which inhibited cholesterol biosynthesis by greater than 85% and decreased total cell cholesterol from 36.1 to 27.4 micrograms/ml of cell protein. Mevinolin treatment caused a 4.9 +/- 0.8-fold increase in the amount of H-TGL activity secreted into the medium, a 1.8 +/- 0.4-fold rise in H-TGL-specific mRNA, and a concurrent 14-fold increase in HMG-CoA reductase mRNA. Addition of 1 mM mevalonic acid to normal or mevinolin-treated cells raised the cellular cholesterol content and decreased the amount of secreted H-TGL activity to levels below control values. Mevalonic acid also prevented mevinolin-induction of H-TGL and HMG-CoA reductase mRNA, suggesting a common regulatory step for H-TGL and HMG-CoA reductase. Exposure of cells to mevinolin and 25-hydroxycholesterol together resulted in a marked repression of HMG-CoA reductase mRNA levels, whereas these conditions further enhanced the secretion of H-TGL activity and the expression of H-TGL mRNA. These results demonstrate a differential role for 25-hydroxycholesterol in the regulation of H-TGL and HMG-CoA reductase expression.

Characterization and sequence analysis of the human ornithine decarboxylase gene.

This report describes the characterization and complete sequence of the human ornithine decarboxylase (ODC) gene. Genomic Southern blot analysis shows only a single gene hybridizing at high stringency, in contrast to the murine multigene family. A Pst I restriction fragment length polymorphism was identified and an allele of the human ODC gene containing the polymorphic Pst I site was cloned and sequenced. The ODC gene is divided into 12 exons and spans 8 kb. Comparison of the human, rat, and mouse ODC genes shows striking conservation of genomic organization, as well as 82% identity in the first 148 bp of the 5'-flanking region. This region contains a TATA box, cAMP-responsive element, CCAAT box, and AP-2 binding site and is consistent with induction of ODC gene expression by both the cAMP and protein kinase C-mediated signaling pathways. The first intron of the human gene is 2,849 bp in length, and contains two putative Sp1 binding sites, as well as an Ap1 binding site, suggesting a role for the first intron in transcriptional regulation. The 5' noncoding region of the predicted mRNA contains regions of virtual identity with that of mouse and rat ODC mRNA, suggesting sequences involved in translational regulation. In addition, it was found that the exon segments corresponding to the amino and carboxyl termini of Saccharomyces cerevisiae and Trypanosoma b. brucei are unrelated to their mammalian counterparts, whereas the middle segments of the protein are conserved. These differences may influence the difference in protein half-life seen between T. b. brucei and mammalian ODC.

pH-dependent processes in proteins.

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.

Contributions of individual amino acid residues to the structural stability of cetacean myoglobins.

The acid-denaturation behavior of eleven cetacean myoglobins has been studied at two ionic strengths, 0.01 and 0.10 M, at 25.0 degrees C. The myoglobulins studied fall into four phylogenetic suborders, representing the sperm whales, dolphins, baleen whales, and beaked whales. The differences in response to acid denaturation among these closely related myoglobins are small but statistically significant. In three cases, free-energy differences between myoglobins can be ascribed to one amino acid difference and in three others to two differences. The differences in response were analyzed in terms of the changes in noncovalent interactions occurring in the native structure. The effects of changes in electrostatic interactions over the whole charge array were calculated for each myoglobin species by using the modified Tanford-Kirkwood theory. The predicted changes in stability correlated well with the experimental observations in most cases. When differences in hydrogen-bonding capability were considered at a first approximation, substantial effects were predicted. When these effects were taken in conjunction with the electrostatic interactions, the correlation with experiment was improved. Additionally, restrictions in motional freedom and packing constraints appeared to be significant in the single-site analysis. The detectable differences in stability due to single amino acid substitutions along with the small differences in stability between the cetacean suborders indicate that compensatory interactions provide the mechanism for the conservation of stability among the myoglobins studied.

Electrostatic contributions to the energetics of dimer-tetramer assembly in human hemoglobin: pH dependence and effect of specifically bound chloride ions.

The pH dependence and effects of specifically bound chloride ions on the electrostatic contribution to the energetics of human hemoglobin dimer-tetramer assembly were computed for deoxy- and liganded hemoglobin. In the absence of bound chloride, the electrostatic contribution models the observed contrasting pH dependence of dimer-tetramer assembly for deoxy- and oxyhemoglobin. The effect of specifically bound chloride on the computations depends on the number and placement of the anions. Deoxy assembly shows a greater sensitivity to anion binding, with effects propagating as far as 32 A from the binding site. This sensitivity suggests a mechanism for electronic communication with the heme. At pH 7.4, 24-34% of the experimental value for deoxy and 73-85% for oxy dimer-tetramer assembly stabilization are predicted. Together with the findings of Chu and Ackers [Chu, A. H., & Ackers, G. K. (1981) J. Biol. Chem. 256, 1199] and other recent work, these results suggest that salt bridge formation is not the dominant energetic factor favoring deoxyhemoglobin dimer-tetramer assembly. Results of this work suggest that the marked electrostatic stabilization favoring oxy dimer-tetramer assembly may be a significant contributor to the quaternary enhancement observed in assembly reactions whereas the nonelectrostatic factors favoring deoxy dimer-tetramer assembly may be largely responsible for quaternary constraint.