Functional and structural basis of carnitine palmitoyltransferase 1A deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.     

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.     

Association study between interleukin-1beta gene (IL-1beta) and schizophrenia

An increasing amount of evidence suggests that the pathophysiology of schizophrenia is associated with the abnormal immune system, and cytokines may be important in schizophrenia. Among these cytokines, interleukin-1beta may play a role in the pathogenesis of the disease. In the present study, we investigated the genetic association between a TaqI polymorphism in interleukin-1beta gene (IL-1beta) and schizophrenia by restriction fragment length polymorphism (RFLP) analysis among 132 Chinese families of Han descent. The transmission disequilibrium test (TDT) did not demonstrate an allelic association with schizophrenia. Our results suggested that the TaqI polymorphism in IL-1beta gene might not confer increased susceptibility for schizophrenia.     

Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erbB2 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors.     

Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction

We examined ultraviolet (UV) irradiation and cisplatin treatment damage formation and repair efficiency in the p53 tumor suppressor gene of various cultured cell lines and lymphocytes using a nonradioactive multiplex long quantitative polymerase chain reaction (QPCR) assay, which amplified a 7-kb fragment of the target gene and a 500-bp fragment of the template control to successfully increase the sensitivity and reliability of the assay. The multiplex long QPCR detected a lesion frequency of 0.63 lesions/10kb/10J/m(2) in the p53 gene of fibroblast cells. In addition, the multiplex long QPCR assay detected pronounced differences in the repair of UV damage in the p53 gene among repair-proficient CRL-1475 cells and repair-deficient XP-A and XP-C cells. The multiplex long QPCR assay was also evaluated as a sensitive assay for the detection of DNA damage induced by cisplatin. The data indicated that the lesion frequency in the p53 gene was 1.27-1.75 times higher in the H23 cisplatin-sensitive cell than in the H1435 cisplatin-resistant cell at the IC(70) dose. After 8-h and 24-h repair periods, only 13 and 75% of cisplatin-induced damage had been removed in the H23 cells, whereas these values were 92 and 100% in the H1435 cells. In addition, our data indicate that multiplex long QPCR is a sensitive method for validly estimating repair in freshly isolated lymphocytes. The results suggest that the current protocol of the multiplex long QPCR method can be used to assess the damage formation and repair efficiency of various agents at biologically relevant doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in epidemiological studies.     

Crystal structure of a NO-forming nitrite reductase mutant: an analog of a transition state in enzymatic reaction

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.