Patterns of importin-alpha expression during Drosophila spermatogenesis

Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, -alpha2, and -alpha3. We found that each Imp-alpha is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.     

Using mouse models to dissect the genetics of obesity

Mice have proved to be powerful models for understanding obesity in humans and farm animals. Single-gene mutants and genetically modified mice have been used successfully to discover genes and pathways that can regulate body weight. For polygenic obesity, the most common pattern of inheritance, many quantitative trait loci (QTLs) have been mapped in crosses between selected and inbred mouse lines. Most QTL effects are additive, and diet, age and gender modify the genetic effects. Congenic, recombinant inbred, advanced intercross, and chromosome substitution strains are needed to map QTLs finely, to identify the genes underlying the traits, and to examine interactions between them.     

Covalent incorporation of selenium into oligonucleotides for X-ray crystal structure determination via MAD: proof of principle. Multiwavelength anomalous dispersion

Selenium was incorporated into an oligodeoxynucleotide in the form of 2'-methylseleno-uridine (U(Se)). The X-ray crystal structure of the duplex left open bracket d(GCGTA)U(Se)d(ACGC) right open bracket (2) was determined by the multiwavelength anomalous dispersion (MAD) technique and refined to a resolution of 1.3 A, demonstrating that selenium can selectively substitute oxygen in DNA and that the resulting compounds are chemically stable. Since derivatization at the 2'-alpha-position with selenium does not affect the preference of the sugar for the C3'-endo conformation, this strategy is suitable for incorporating selenium into RNA. The availability of selenium-containing nucleic acids for crystallographic phasing offers an attractive alternative to the commonly used halogenated pyrimidines.     

Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1)

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.     

Advanced glycation end-product (AGE)-damaged IgG and IgM autoantibodies to IgG-AGE in patients with early synovitis

Advanced glycation end-product (AGE)-damaged IgG occurs as a result of hyperglycemia and/or oxidative stress. Autoantibodies to IgG-AGE were previously demonstrated in patients with severe, longstanding rheumatoid arthritis (RA). We investigated whether IgG-AGE and anti-IgG-AGE antibodies were present early in the course of RA and other inflammatory arthropathies. We prospectively followed a cohort of 238 patients with inflammatory arthritis of duration less than 1 year. Patients were evaluated clinically and serologically, and radiographs were obtained at initial and 1-year visits. Sera were assayed for IgG-AGE and anti-IgG-AGE antibodies by enzyme-linked immunosorbent assay (ELISA). Rheumatoid factor (RF) was determined by nephelometry and ELISA. Of all patients, 29% had RF-positive RA, 15% had RF-negative RA, 18% had spondyloarthropathy, and 38% had undifferentiated arthritis. IgG-AGE was present in 19% of patients, and was similar in amount and frequency in all groups. Patients with elevated IgG-AGE levels had significantly higher levels of the inflammatory markers C-reactive protein and erythrocyte sedimentation rate, but there was no correlation with blood glucose levels. Overall, 27% of the patients had IgM anti-IgG-AGE antibodies. These antibodies were highly significantly associated with RFs (P < 0.0001) and with swollen joint count (P < 0.01). In early onset arthritis, IgG damaged by AGE was detected in all patient groups. The ability to make IgM anti-IgG-AGE antibodies, however, was restricted to a subset of RF-positive RA patients with more active disease. The persistence of the anti-IgG-AGE response was more specific to RA, and was transient in the patients with spondyloarthropathy and with undifferentiated arthritis who were initially found to be positive for anti-IgG-AGE antibodies.     

Perioperative Myocardial Injury after Adult Heart Transplant: Determinants and Prognostic Value

MethodsData on 362 consecutive recipients (mean age: 47.8±13.7, 20.2% female, 18.2% diabetics, 22.1% with previous cardiac operations, 27.6% hospitalized, 84.9±29.4 ml/min preoperative glomerular filtration rate) were analyzed using multivariable logistic regression modeling. Target outcomes were determinants of troponin release, early graft failure (EGF), acute kidney injury (AKI) and operative death.ResultsMean cTnI release measured 24 hours after transplant was 10.9±11.6 μg/L. Overall hospital mortality was 10.8%, EGF 10.5%, and AKI was 12.2%. cTnI release>10 μg/L proved an independent predictor of EGF (OR 2.2; 95% CI, 1.06–4.6) and AKI (OR 1.031; 95% CI, 1.001-1.064). EGF, in turn, proved a determinant of hospital mortality. Risk factors for cTnI>10 μg/L release were: status 2B (OR 0.35; 95% CI, 0.18-0.69, protective), duration of the ischemic period (OR 1.006; 95% CI, 1.001-1.011), previous cardiac operation (OR 2.9; 95% CI, 1.67-5.0), and left ventricular hypertrophy (OR 3.3; 95% CI, 1.9-5.6).ConclusionsMyocardial enzyme leakage clearly emerged as an epiphenomenon of more complicated clinical course. The complex interplay between surgical procedure features, graft characteristics and recipient end-organ function highlights cTnI release as a risk marker of graft failure and acute kidney injury. The search for optimal myocardial preservation is still an issue.