Divergent Reactivity of Hemoglobin Isoforms α2Aß2 and α2Dß2 from Meleagris gallopavo in Native and Fatty Acid-Modified States.

Meleagris gallopavo (turkey) coexpresses distinct hemoglobin (Hb) isoforms, Hb α2Aß2 (HbA) and α2Dß2 (HbD), at a ratio of ∼3:1 (HbA:HbD). Herein, the reactivities of HbA and HbD were investigated in their native and free fatty acid (FFA)-modified states. Results indicated that HbD displays elevated autoxidation (kox) and an increased propensity to oxidize lipids in its reduced (oxy) and oxidized (met) forms. Interestingly, metHbD displayed less heme-globin cross-linking compared to HbA. Regarding FFA-modified Hb, we found that an FFA mixture and linoleic acid (LA) produced a bis-histidyl ferric (Bis-His) Hb species, decreasing the ability of Hb to oxidize lipids. Using molecular docking, we found LA to hydrogen bond with ß Arg C6, found at the α1ß2 interface, but the extent of Bis-His formation differs between HbA and HbD. Our findings suggest HbA displays elevated oxidative stability compared to HbD and that FFA may act as allosteric effectors of metHb.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

In vitro biomechanical comparison of 3.5 mm LC-DCP/intramedullary rod and 5 mm clamp-rod internal fixator (CRIF)/intramedullary rod fixation in a canine femoral gap model.

OBJECTIVE: To compare the biomechanical properties of clamp rod internal fixation (CRIF)/rod and LC-DCP/rod constructs in a canine femoral gap model. STUDY DESIGN: Cadaveric biomechanical study. SAMPLE POPULATION: Canine femora (n = 10 pair). METHODS: Femora with 40 mm ostectomies were assigned to LC-DCP/rod or CRIF/rod treatment groups. Five construct pairs had 4-point bending and 5 pairs had torsional loading. Construct stiffness, strength, and bending angle at failure or permanent angular deformation (torsional loading) were determined. Statistical comparisons were performed using Student t tests; significance was set at P ≤ .05. RESULTS: There was significantly greater permanent angular deformation, or residual twist, in the CRIF/rod constructs (23.1 ± 0.89°) compared with LC-DCP/rod constructs (7.47 ± 2.08°). Whereas there was no significant difference in torsional stiffness of these constructs at torsional loads <4.92 N m (P = .819), LC-DCP/rod constructs had significantly greater torsional stiffness (0.303 ± 0.079 N m/°) and strength (11.546 ± 2.79 N m) than CRIF/rod construct stiffness (0.06 ± 0.013 N m/°) and strength (6.078 ± 0.527 N m) at torsional loads >4.92 N m. Differences in stiffness and strength in 4-point bending were not statistically significant. CONCLUSIONS: LC-DCP/rod constructs had significantly less permanent angular deformation than CRIF/rod constructs. CRIF/rod constructs became less stiff as torsional load was increased, thus the LC-DCP/rod constructs had significantly greater torsional stiffness and strength under high torsional loads. LC-DCP/rod and CRIF/rod constructs performed similarly under 4-point bend loading conditions.

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.