Analysis of mouse skin reveals proteins that are altered in a diet-induced diabetic state: a new method for detection of type 2 diabetes

In this study, proteomic analysis was performed on the skin of C57BL/6J mice with type 2 diabetes and compared to nondiabetic controls. To induce obesity and subsequent diabetes, mice were placed on a high-fat diet for 16 wk. After 16 wk, both diabetic and nondiabetic control mice were sacrificed and their skin removed for analysis. Following 2-DE, proteomic profiles from the skin samples were quantified using PDQuest software. Out of more than 1000 distinct protein spots, 28 were shown to be significantly altered with 6 being decreased and 22 increased in the diabetic state compared to controls. The 28 protein spots were removed from the gels and analyzed by MALDI-TOF and MS/MS analyses. Protein identifications revealed that 17 of the 28 proteins were involved in energy metabolism (60.7% of changes observed). Collectively, none of the significantly altered proteins had been shown previously to be altered in diabetic skin. This study not only helps to identify proteins found in skin samples of obese mice with type 2 diabetes, but also shows that skin biopsies coupled with proteomic analysis may be useful as a noninvasive method for the diagnosis of hyperinsulinemia and diabetes.     

Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

Crystallization and preliminary data of Indian buffalo erythrocyte carbonic anhydrase

The most abundant anhydrase isoenzyme from the erythrocyte of Indian buffalo has been purified using affinity gel and DEAE-cellulose ion-exchange columns and single crystals suitable for X-ray diffraction studies have been obtained. The unit cell dimensions are a = 46.8 A, b = 104.5 A, c = 60.4 A, beta = 91.2 degrees and the space group is P2(1), with two molecules per asymmetric unit.     

A unique and essential role for insulin in the phenotypic expression of rat mammary epithelial cells unrelated to its function in cell maintenance

Mammary explants from pregnant rats can be induced in regard to casein synthesis and alpha-lactalbumin activity when cultured in the presence of hydrocortisone, prolactin and levels of insulin approaching physiological concentrations. No detectable induction occurs in the absence of insulin. Although epidermal growth factor and multiplication stimulating activity, in the presence of hydrocortisone, can maintain the initial level of NADH-cytochrome c reductase as well as insulin, neither can substitute effectively for insulin in the induction of the milk proteins. Proinsulin, nerve growth factor, platelet-derived growth factor and fibroblast growth factor are also ineffective substitutes for insulin in this regard. Whereas prolonged tissue exposure to multiplication stimulating activity, hydrocortisone and prolactin does not result in induction of alpha-lactalbumin activity, subsequent addition of insulin leads to prompt response. The results suggest that the ability of insulin to function as a unique, essential factor in the induction of rat milk proteins is independent of its cell-maintenance activity. Thus, in addition to its well established functions in metabolic processes, insulin appears to play a vital role in certain developmental processes.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of β-ketoacyl ACP synthase I bound to platencin and fragment screening molecules at two substrate binding sites

The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The β-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the β-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol. The enzyme is a dimer and based on structural and sequence conservation, harbors the same active site configuration as other β-ketoacyl-ACP synthases. The platencin binding site overlaps with the fatty acyl compound supplied by ACP, while 7-hydroxyl-coumarin and (5-thiophen-2-ylisoxazol-3-yl)methanol bind at the secondary fatty acyl binding site. These high-resolution structures, ranging between 1.25 and 1.70 å resolution, provide a basis for in silico inhibitor screening and optimization, and can aid in rational drug design by revealing the high-resolution binding interfaces of molecules at the malonyl-ACP and acyl-ACP active sites.     

Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.