Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Eradication of Helicobacter pylori for the prevention of peptic ulcer rebleeding

AIM: To evaluate the effect of Helicobacter pylori eradication on ulcer bleeding recurrence in a prospective, long-term study including more than 400 patients. METHODS: Patients with peptic ulcer bleeding were prospectively included. H. pylori infection was confirmed by rapid urease test, histology or (13)C-urea breath test. Several eradication regimens were used. Ranitidine 150 mg was administered daily until eradication was confirmed by breath test 8 weeks after completing eradication therapy. Patients with therapy failure received a second or third course of therapy. Patients with eradication success did not receive maintenance anti-ulcer therapy, and were controlled yearly with a repeated breath test. RESULTS: Four hundred and twenty-two patients were followed up for at least 12 months, with a total of 906 patient-years of follow up. Mean age was 59 years, and 35% were previous nonsteroidal anti-inflammatory drug (NSAID) users. Sixty-nine percent had duodenal, 24% gastric, and 7% pyloric ulcer. Recurrence of bleeding was demonstrated in two patients at 1 year (incidence: 0.22% per patient-year of follow up), which occurred after NSAID use in both cases. CONCLUSION: Peptic ulcer rebleeding does not occur in patients with complicated ulcers after H. pylori eradication. Maintenance anti-ulcer (antisecretory) therapy is not necessary if eradication is achieved.     

The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).     

Cytotoxicity mechanisms of pyrazino[1,2-b]isoquinoline-4-ones and SAR studies

The cytotoxicity showed by 1b, an interesting representant of the title compounds, for HT-29 human colon cancer cells (CI₅₀ value of 1.95 × 10^?7 M) has been related to the induced cell death at the G2 phase and not to DNA damage. This compound promotes the degradation of components of the G2/M checkpoint machinery, in particular cdc2, Cyclin B1 and Wee1, which represents a novel mechanism of cytotoxicity. Degradation of Wee1 seems to be mediated by proteasome activity but degradation of cdc2 has to occur through a different mechanism. The activity of 1b on G2 cell cycle components suggests that tumor cells that are arrested in G2/M by anticancer drugs like cisplatin could be targeted by compound 1b, increasing the apoptosis induction, and that their optimized analogs might be useful in the treatment of colon cancer through combination therapies with cisplatin or other anticancer drugs that affect the cytoskeleton integrity such as taxol and taxotere.SAR studies with compounds obtained by manipulation of the N(2) and C(4)-functional groups and the C(6)-chain of compound 1b have confirmed the importance of these structural features in the in vitro antitumor activity. Fused oxazolidine derivatives as compound 5 were inactive, and the lack of activity found in the replacement of the C(4)-lactam by a cyanoamine function, as in compounds 8–10, could be explained considering that their all-syn relative configuration makes them too stable to generate alkylating iminium species.     

The alpha chains of goat hemoglobins: old and new variants in native Apulian breeds

Blood samples were collected from 324 goats belonging to the native Apulian breeds Garganica and Jonica; 60 Alpine goats were also sampled to serve as a comparison. Hemoglobin phenotypes were analyzed with isoelectric focusing in a pH range of 6.7-7.7. Heterogeneity of globin chains was evidenced both by AUT-PAGE and RP-HPLC. The primary structure of four alpha globins was analyzed by combined mass spectrometry approaches. Two of these globins had never been sequenced before. One was a new alpha variant, an allele of the HBA1A gene from which it differed for the mutation A26T and has been registered with a low frequency only in Apulian breeds; the other was a globin encoded by the HBA2 locus, whose primary structure was previously derived from the corresponding gene. The two alleles recorded at the HBA2 locus presented a different frequency in the three breeds but may be considered to be generally rather common. Notwithstanding the sample size no goat was found to exhibit HbA1B. The authors discuss their findings in the light of the results reported by other researchers and argue that, in spite of what had been inferred in pioneer works on goat hemoglobins, HBA1B is not a common allele.