The NR4A family of orphan nuclear receptors are not required for adipogenesis.

Nuclear hormone receptors of the NR4A subfamily are rapidly induced during the early stages of adipogenesis, leading to the speculation that they may have important roles in this process. One of the three subfamily members, Nur77 has also been shown to play key roles in energy expenditure and lipolysis in skeletal muscle and in the control of hepatic gluconeogenesis. We, therefore, examined the role of NR4A factors in adipogenesis using the well-characterized 3T3-L1 preadipocyte model. Inhibition of Nur77 expression using siRNA did not affect induction of adipogenic genes, nor the accumulation of lipid. To inhibit the activity of all the three NR4A family members, we generated preadipocytes stably expressing a well-characterized dominant-negative Nur77 (DN-Nur77), known to block the function of the other NR4A factors, Nurr1 and Nor1, as well as Nur77. While the increased NR4A activity observed following adipogenic induction was completely abolished in these cells, DN-Nur77 expression did not affect the expression of genes characteristic of terminally differentiated adipocytes and had no impact on lipid accumulation in these cells. Thus, while members of the NR4A subfamily of nuclear receptors may have important metabolic roles in skeletal muscle and liver, we demonstrate that they are dispensable for normal adipocyte development.

Homology modeling and substrate binding study of human CYP2C9 enzyme.

The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.

Homology modeling and substrate binding study of human CYP2C18 and CYP2C19 enzymes.

It is well established that the variable binding-site architecture and composition of the P450 metabolizing heme proteins are major modulators of substrate and product specificity. Even the three closely related human liver isozymes, CYP2C9, CYP2C18, and CYP2C19, do not share all substrates and do not always lead to the same preferred hydroxylation products. The lack of knowledge of their three-dimensional (3D) structures has hindered efforts to understand the differences in their specificities. Building on previous work for the CYP2C9 enzyme, 3D models of CYP2C18 and 2C19 have been constructed and validated by computational methods developed and tested in our laboratory. They were used to characterize explicit enzyme-substrate complexes using the isoform-specific substrates progesterone and (S)-mephenytoin for 2C19 and 2-[2,3-dichloro-4-(3-hydroxypropyloxy)benzoyl]thiophene for 2C18. The results allowed both common and unique binding-site residues to be identified in each model. The calculated preferred hydroxylation site was obtained for each substrate and was found to be consistent with experimental observation. Comparisons were made among the 2C9, 2C18, and 2C19 model binding sites to investigate the subtle differences among them. These models can be used as structure-based guides for mutagenesis studies and screening of potential pharmaceuticals or toxins.

Protein hydration and unfolding--insights from experimental partial specific volumes and unfolded protein models.

BACKGROUND: The partial specific volume of a protein is an experimental quantity containing information about solute-solvent interactions and protein hydration. We use a hydration-shell model to partition the partial specific volume into an intrinsic volume occupied by the protein and a change in the volume occupied by the solvent resulting from the solvent interactions with the protein. We seek to extract microscopic information about protein hydration and unfolding from experimental volume measurements without using computer simulations. We employ the idea that the protein-solvent interaction will be proportional to the surface area of the protein. RESULTS: A linear relationship is obtained when the difference between the experimental protein partial specific volume and its intrinsic volume is plotted as a function of the protein solvent-accessible surface area. The effect of using different protein volume definitions on the analysis of protein volumetric properties is discussed. Volumetric data are used to test a model for the unfolded state of proteins and to make predictions about the denatured state. CONCLUSIONS: The linear relationship between hydration-shell volume change and accessible surface area reflects the similar surface properties (fractional composition of nonpolar, polar and charged surface) among a diverse set of proteins. This linear relationship is found to be independent of how the solution is partitioned into solute and solvent components. The interpretation of hydration shell versus bulk water properties is found to be very model dependent, however. The maximally exposed unfolded protein model is found to be inconsistent with experimental volume changes of unfolding.

Escherichia coli in patients with acute gastroenteritis in Lagos, Nigeria.

Over a period of 12 months, a total of 852 stool samples from patients (both children and adults) with acute diarrhoeal diseases attending some public and government recognised health institutions in Lagos metropolis were screened for diarrhoeagenic bacterial agents. One hundred and eighty two bacterial isolates were found out of which 83 (45.6%) were Escherichia coli, 38 (20.9%) Shigella spp, 31 (17%) Salmonella spp, 16 (8.8%) Klebsiella spp, eight (4.4%) Aeromonas spp, while there were only six (3.3%) isolates for Plesiomonas spp. Of the 83 isolates for E. coli group, 49 (59%) were enteropathogenic (EPEC), 17 (20.5%) enterotoxigenic (ETEC), 10 (12.1%) enteroinvasive (EIEC) and seven (8.4%) enterohaemorrhagic (EHEC). The EPEC strains particularly serotype 055, were mostly encountered in children aged over five years. On the other hand, EIEC and ETEC strains were found mainly in adults while EHEC O157: H7 strains occurred in all the age groups studied. This study further stresses the important role of EIEC and ETEC in acute diarrhoeal diseases and the possible implication of EHEC in acute gastroenteritis, especially in children in Lagos, Nigeria.