Red cell membrane lipid peroxidation in transfusion-dependent B and EB thalassemia

Red cell membrane lipid peroxidation in B and EB thalassemias has been found to increase significantly. The increased iron status of the patients, as revealed by the serum ferritin level, did not have any correlation with lipid peroxidation in either of the groups.     

Effect of acute hypoxia on glomus cell Em and psi m as measured by fluorescence imaging.

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.     

A new approach for modification of phenylalanine peptides by Suzuki-Miyaura coupling reaction

For the first time, we have modified phenylalanine peptides by the Suzuki-Miyaura coupling reaction which may be useful in developing combinatorial libraries of peptidomimetics.     

In vitro perfused-superfused cat carotid body for physiological and pharmacological studies

An in vitro perfused carotid body preparation was developed to study its chemosensory responses to physiological and pharmacological stimuli. The carotid bifurcation with the carotid body was vascularly isolated and excised from pentobarbital sodium-anesthetized cats. The CB was perfused in a chamber by gravity (80 Torr) with modified Tyrode's solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH at pH 7.40) equilibrated at a given Po2 and superfused with the same medium at (Po2 of 20 Torr). The temperature was maintained at 35.5 +/- 0.5 degrees C. The frequency of chemosensory discharges (CD) was recorded from the whole carotid sinus nerve (n = 24), and the responses were tested by repeated interruptions of perfusate flow (SF), perfusion with hypoxic medium, and injections of nicotine and cyanide (0.1 nmol to 1 mumol) and hypercapnic medium. During hyperoxic perfusion, SF resulted in a sigmoidal increase in CD, reaching a maximum that was 23.6 +/- 4.4-fold greater than the basal activity. Restoration of flow returned CD promptly to basal values. After normoxic perfusion, SF led to a similar maximal activity more rapidly, but the duration was shorter. Reduction of the perfusate PO2 (Po2 from 450 Torr to 150, 30, and less than 10 Torr) caused a nonlinear increase in CD. CO2 stimuli (PCo2 38-110 Torr) resulted in a linear increase in CD. Nicotine or cyanide increased CD in a dose-dependent manner. The preparation retained its initial responsiveness for 2-3 h, making extensive experimental studies feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein

Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins.     

MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed

The full repertoire of regulatory interactions utilized by human cells to control expression of amyloid-β precursor protein (APP) is still undefined. We investigated here the contribution of microRNA (miRNA) to this regulatory network. Several bioinformatic algorithms predicted miR-101 target sites within the APP 3′-untranslated region (3′-UTR). Using reporter assays, we confirmed that, in human cell cultures, miR-101 significantly reduced the expression of a reporter under control of APP 3′-UTR. Mutation of predicted site 1, but not site 2, eliminated this reporter response. Delivery of miR-101 directly to human HeLa cells significantly reduced APP levels and this effect was eliminated by co-transfection with a miR-101 antisense inhibitor. Delivery of a specific target protector designed to blockade the interaction between miR-101 and its functional target site within APP 3′-UTR enhanced APP levels in HeLa. Therefore, endogenous miR-101 regulates expression of APP in human cells via a specific site located within its 3′-UTR. Finally, we demonstrate that, across a series of human cell lines, highest expression of miR-101 levels was observed in model NT2 neurons.