Biomedical subjects

H Razzaghi

Publications and source records attributed to H Razzaghi.

5 recordsLinked to original sources

PubMed2001

A highly sensitive and nonradioactive mutation detection method based on vertical gradient temperature single-strand conformation polymorphism.

The single-strand conformation polymorphism (SSCP) method is widely used for mutation detection. The sensitivity of the method depends on several factors, most importantly on the temperature at which electrophoresis of single-stranded DNA (ssDNA) takes place. The temperature has a profound effect on the folded conformation of ssDNA. The temperature factor is predominantly determined empirically in conventional SSCP, which can be very tedious especially when a large number of different DNA samples need to be screened. We have devised a novel SSCP method based on a vertical gradient temperature (VGT), which automatically subjects ssDNA to various temperatures in the same electrophoresis. The theory behind VGT-SSCP protocol is that when ssDNA is subjected to run in a wide range of gradient temperature, it will automatically acquire optimal resolution at an optimal temperature to distinguish between the wild type and the mutant type ssDNA. The sensitivity level of mutation detection of VGT-SSCP depends on whether the corresponding optimal secondary structure of a mutant DNA strand is within the preset gradient temperature range. In summary, the VGT-SSCP is a simple and robust nonradioactive method that is more sensitive than constant-temperature SSCP in detecting unknown mutations.

DNA, Single-Stranded↗

PubMed2001

Structure-function analysis of D9N and N291S mutations in human lipoprotein lipase using molecular modelling.

Lipoprotein lipase (LPL) plays a central role in lipid metabolism. The D9N and N291S mutations in the LPL gene are associated with elevated triglyceride and decreased HDL-cholesterol levels. Published in vitro expression studies suggest that these two mutations are associated with reduced LPL enzymatic activity. We sought to gain further insight on the impact of these two mutations on the LPL structure and function by molecular modelling techniques. Homology modelling was used to develop a three-dimensional (3D) structure of LPL from human pancreatic lipase. Two separate LPL models for the D9N and N291S substitutions were constructed and compared with the wild type LPL for differences in hydrophobicity, atomic burial, hydrogen bond pattern, and atomic mobility. In comparison to the wild type model, the 9N model was associated with significantly increased atomic mobility of its neighboring residues, but the catalytic site was not affected. The region near residue 9 in the upper part of the N-domain was considered a candidate site for protein-protein interaction. In the N291S model, alterations in H-bonds and constrained atomic mobility were among conformational changes in the region where the substitution had occurred. These are hypothesized to cause an increase in the rate of dissociation in LPL dimerization, subsequently affecting the LPL enzymatic activity. We also modelled the C-domain of apoCII, the obligatory cofactor of LPL, from 2D NMR data and docked the model with LPL to explore their interaction site. These docking experiments suggest that the C-domain of apoCII interacts with the interface of N- and C-domains of LPL and part of the lid structure that covers the catalytic site. In summary, we provide molecular modelling data on two well-known mutations in the LPL gene to help explain the published in vitro expression findings and propose a possible LPL-apoCII interaction site. Our data indicate that molecular modelling of LPL mutations could provide a valuable tool to understand the effects of a mutation on the structure-function of this important enzyme.

Amino Acid Sequence↗

PubMed2001

The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.

3T3 Cells↗

PubMed2000

Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels.

The lipoprotein lipase (LPL) enzyme plays a major role in lipid metabolism, primarily by regulating the catabolism of triglyceride (TG)-rich lipoprotein particles. The gene for LPL is an important candidate for affecting the risk of atherlosclerosis in the general population. Previously, we have shown that the HindIII polymorphism in intron 8 of the LPL gene is associated with plasma TG and HDL-cholesterol variation in Hispanics and non-Hispanic whites (NHWs). However, this polymorphism is located in an intron and hence may be in linkage disequilibrium with a functional mutation in the coding region or intron-exon junctions of the LPL gene. The aim of this study was to initially screen the LPL coding region and the intron-exon junctions by single-strand conformation polymorphism (SSCP) analysis for mutation detection in a group of 86 individuals expressing the phenotype of high TG/low HDL, followed by association studies in a population-based sample of 1,014 Hispanics and NHWs. Four sequence variations were identified by SSCP and DNA sequencing in the coding region of the gene, including two missense mutations (D9N in exon 2 and N291S in exon 6), one samesense mutation (V108V in exon 3), and one nonsense mutation (S447X in exon 9). Multiple regression analyses, including these four mutations and the HindIII polymorphic site, indicate that the association of the HindIII site with plasma TG (P=0.001 in NHWs and P=0.002 in Hispanics) and HDL-cholesterol (P=0.007 in NHWs and P=0.127 in Hispanics) is independent of all other LPL variable sites examined. These observations reinforce the concept that the intronic 8 HindIII site is functional by itself and provide a strong rationale for future comprehensive functional studies to delineate its biological significance.

Adult↗

PubMed1996

Correct cell- and differentiation-specific expression of a murine alpha 1 (I) collagen minigene in vitro differentiating embryonal carcinoma cells.

An in vitro differentiation system utilizing retinoic acid (RA) treatment of pluripotent murine P19 embryonal carcinoma (EC) cells, which can be induced to differentiate into various cell types, was optimized for maximal induction of alpha 1 type I collagen (Col1a1) gene expression. Differentiation was associated with apoptotic death of the majority of cells, indicating that this in vitro system faithfully mimics the in vivo differentiation process. Col1a1 mRNA became detectable by RNase protection assay after 3 days of RA treatment and, after 6 days, reached a level comparable to that in NIH 3T3 fibroblasts. After induction of differentiation the Col1a1 gene remained transcriptionally active for extended periods of time even in the absence of RA. A minigene version of the murine Col1a1 gene was constructed that contains all of the so far known Col1a1 regulatory elements. This construct exhibited the correct expression pattern in stable transfection experiments: it was expressed in fibroblasts, but not in undifferentiated P19 EC cells, and it was transcriptionally activated after induction of differentiation. This experimental system should be a useful tool for dissecting the molecular mechanisms involved in the developmental activation and stage- and tissue-specific expression of the murine Col1a1 gene.

3T3 Cells↗