Vitamin E or coenzyme Q10 administration is not fully advantageous for heart mitochondrial function in diabetic goto kakizaki rats

The heart is one of the organs affected during the later stages of diabetes. Mitochondrial function has already been proposed to be affected during the course of diabetes. Nevertheless, little information is known concerning the impact of antioxidants in heart mitochondria of a milder model for diabetes, such as the Goto-Kakizaki (GK) rat, where mitochondrial function appears ameliorated. The objective of this work was to test if injections of Vitamin E and Coenzyme Q10, alone and in combination, were able to modify mitochondrial performance in the hearts of GK rats. Several aspects of mitochondrial function were measured, such as the respiratory control ratio and the electric potential, as well as the mitochondrial accumulation of Vitamin E and Coenzymes Q9 and Q10. We observed that only Vitamin E appeared to have a positive impact on the mitochondrial phosphorylation efficiency and on mitochondrial performance, namely on the ability to generate the electric transmembrane potential in the presence of supra-physiological calcium concentrations. Vitamin E administration also increased the mitochondrial concentration of Coenzyme Q10. None of the treatments was able to reverse the diabetic phenotype in GK rats. We conclude that in this model of mild hyperglycemia, administration of antioxidants may have a marginal positive impact on mitochondrial function.     

Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2

The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.     

Enantiospecific syntheses of C(5)-functionalized precursors of artemisinin derivatives

Enantiomerically pure compounds as precursors for the synthesis of hydroxylated derivatives of artemisinin/arteether have been prepared from (+)-(S)-carvone and (+)-car-2-ene.     

Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.     

Revisiting the mouse mitochondrial DNA sequence

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Conse quently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.     

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.     

De novo balanced translocation (2;10)(q24;q22) associated with mental retardation

We report a case of a reciprocal translocation between the long arms of the 2 and 10 chromosomes observed in a 14-year-old male with mild mental impairment, compulsive and obsessive behavior. The apparently balanced translocation was characterized by fluorescence in situ hybridization and the karyotype was 46, XY, t(2;10)(q24;q22). The way by balanced chromosomal translocations can lead to a disease phenotype are reviewed and discussed.     

Quaternary structure of the neuronal protein NAP-22 in aqueous solution

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.