Nonheme iron in sickle erythrocyte membranes: association with phospholipids and potential role in lipid peroxidation

Previous studies documented the abnormal association of heme and heme proteins with the sickle RBC membrane. We have now examined RBC ghosts and inside-out membranes (IOM) for the presence of nonheme iron as detected by its formation of a colored complex with ferrozine. Sickle ghosts have 33.8 +/- 18.2 nmol nonheme iron/mg membrane protein, and sickle IOM have 4.3 +/- 3.0 nmol/mg. In contrast, normal RBC ghosts and IOM have no detectable nonheme iron. The combination of heme and nonheme iron in sickle IOM averages nine times the amount of membrane- associated iron in normal IOM. Kinetics of the ferrozine reaction show that some of this nonheme iron on IOM reacts slowly and is probably in the form of ferritin, but most (72% +/- 18%) reacts rapidly and is in the form of some other biologic chelate. The latter iron compartment is removed by deferoxamine and by treatment of IOM with phospholipase D, which suggests that it represents an abnormal association of iron with polar head groups of aminophospholipids. The biologic feasibility of such a chelate was demonstrated by using an admixture of iron with model liposomes. Even in the presence of tenfold excess adenosine diphosphate, iron partitions readily into phosphatidylserine liposomes; there is no detectable association with phosphatidylcholine liposomes. To examine the bioavailability of membrane iron, we admixed membranes and t-butylhydroperoxide and found that sickle membranes show a tenfold greater peroxidation response than do normal membranes. This is not due simply to a deficiency of vitamin E, and this is profoundly inhibited by deferoxamine. Thus, while thiol oxidation in sickle membranes previously was shown to correlate with heme iron, the present data suggest that lipid peroxidation is related to nonheme iron. In control studies, we did not find this pathologic association of nonferritin, nonheme iron with IOM prepared from sickle trait, high-reticulocyte, postsplenectomy, or iron-overloaded individuals. These data provide additional support for the concept that iron decompartmentalization is a characteristic of sickle RBCs.     

Depressive symptoms during buprenorphine vs. methadone maintenance: findings from a randomised, controlled trial in opioid dependence

Research suggests that buprenorphine may possess antidepressant activity. The Beck Depression Inventory was completed at baseline and 3 months by heroin dependent subjects receiving either buprenorphine or methadone maintenance as part of a larger, pre-existing, double blind trial conducted by NDARC (Australia). Depressive symptoms improved in all subjects, with no difference between methadone and buprenorphine groups, suggesting no differential benefit on depressive symptoms for buprenorphine compared to methadone.     

Female and male partner age and menotrophin requirements influence pregnancy rates with human menopausal gonadotrophin therapy in combination with intrauterine insemination

This study analyses the influence of female and male patient age and human menopausal gonadotrophin (HMG) requirements on clinical pregnancy rates and live birth rates with ovulation stimulation using HMG in combination with intrauterine insemination (IUI). In this study, 363 consecutive HMG/IUI treatment cycles in 184 patients carried out at a university fertility centre were analysed in a retrospective fashion. The main outcomes measured were clinical pregnancy rates and live birth rates. Increased female partner age (> or = 35) and male partner age (> or = 40) were found to negatively influence pregnancy rates with HMG/ IUI therapy. In addition, this study demonstrated a critical threshold of HMG requirements beyond which pregnancy did not occur. No pregnancies occurred in treatment cycles requiring > 25 ampoules (1875 IU) of menotrophins to achieve follicular maturity, irrespective of patient age. In conclusion, female partner age, male partner age, and HMG requirements all significantly influence pregnancy rates with HMG/IUI therapy.      

The intron 4c allele of the NOS3 gene is associated with ischemic stroke in African Americans

Background  

Ischemic stroke is the most common cause of disability in North America and in addition to the generally accepted risk factors, there is increasing evidence for the potential pathophysiological role of genes. One of these genes, the endothelial nitric oxide synthase gene (NOS3) has been reported as a genetic risk factor for ischemic stroke. To independently confirm and extend the results of these previous reports, we investigated this gene as a risk factor for stroke in an ethnically diverse study population.     

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.     

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu?NLV?RBH?1993?UK) and the Grimsby virus strain (Hu?NLV?Gimsby?1995?UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.     

Age-dependent variation of T1 and T2 relaxation times of adenocarcinoma in mice

The T1 and T2 values of adenocarcinoma EO 771 inoculated into the hind leg of mice are characterized and correlated with the histopathologic state of the tumor. Growth-dependent changes (indicated by a T1 of 630-910 msec and a T2 of 68-185 msec) can be separated into four characteristic phases. The increase in relaxation times in the early phases (A and B) is due to an increasing amount of viable tumor tissue relative to normal muscle tissue. In the later phases (C and D), a decline of the relaxation parameters is observed that is parallel to an increase in the fraction of necrotic tissue. By multiexponential analysis, two relaxation components (indicated by and, respectively) for T1 and T2 and the corresponding fractions alpha 1 and alpha 2 can be observed for both tumor and surrounding muscle tissue. A tissue criterion ("magnetic resonance fingerprint") is defined by a combination of these multiple parameters. This criterion allows separation of not only muscle and tumor tissue but also viable (early state) and necrotic (late state) tumor tissue.