Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

The effect of phenformin and other adenosine triphosphate (ATP)-lowering agents on insulin binding to IM-9 human cultured lymphocytes

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM-9 human cultured lymphocytes. After a 24-hr preincubation, phenformin induced a twofold increase in specific 125I-insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I-insulin binding and energy metabolism in IM-9 cells was investigated. The phenformin-induced increase in insulin binding to IM-9 cells was related to a time- and dose-dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2-deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM-9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.     

Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

Summary Lysine-rich proteinoids in aqueous solution catalyze the formation of peptides from free amino acids and ATP. This catalytic activity is not found in acidic proteinoids, even though the latter contain some basic amino acid. The pH optimum for the synthesis is about 11, but is appreciable below 8 and above 13. Temperature data indicate an optimum at 20°C or above, with little increase in rate to 60°C. Pyrophosphate can be used instead of ATP, with lesser yields resulting. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous amino acid.Proofs should be sent to S.W. Fox, Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, FL 33134     

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction

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Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.     

Single pot sequential crystallization-distillation as a new purification procedure

A new purification procedure exploiting the simultaneous presence of a solid, liquid, and gas phase in a low surface area system is proposed and discussed. The assumptions of vanishingly low diffusion coefficients in the solid phase and that of the presence of a single “effective impurity” allow to plan the sequence of operations starting from the knowledge of just the melting and boiling points of the substance to be purified and of those of the “effective impurity”. Examples and results are presented.