Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Prdm9 and Meiotic Cohesin Proteins Cooperatively Promote DNA Double-Strand Break Formation in Mammalian Spermatocytes

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Molecular determinants of binding between Gly-Leu-Phe-Gly nucleoporins and the nuclear pore complex

The vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N.     

Increased litterfall in tropical forests boosts the transfer of soil CO2 to the atmosphere

Aboveground litter production in forests is likely to increase as a consequence of elevated atmospheric carbon dioxide (CO(2)) concentrations, rising temperatures, and shifting rainfall patterns. As litterfall represents a major flux of carbon from vegetation to soil, changes in litter inputs are likely to have wide-reaching consequences for soil carbon dynamics. Such disturbances to the carbon balance may be particularly important in the tropics because tropical forests store almost 30% of the global soil carbon, making them a critical component of the global carbon cycle; nevertheless, the effects of increasing aboveground litter production on belowground carbon dynamics are poorly understood. We used long-term, large-scale monthly litter removal and addition treatments in a lowland tropical forest to assess the consequences of increased litterfall on belowground CO(2) production. Over the second to the fifth year of treatments, litter addition increased soil respiration more than litter removal decreased it; soil respiration was on average 20% lower in the litter removal and 43% higher in the litter addition treatment compared to the controls but litter addition did not change microbial biomass. We predicted a 9% increase in soil respiration in the litter addition plots, based on the 20% decrease in the litter removal plots and an 11% reduction due to lower fine root biomass in the litter addition plots. The 43% measured increase in soil respiration was therefore 34% higher than predicted and it is possible that this 'extra' CO(2) was a result of priming effects, i.e. stimulation of the decomposition of older soil organic matter by the addition of fresh organic matter. Our results show that increases in aboveground litter production as a result of global change have the potential to cause considerable losses of soil carbon to the atmosphere in tropical forests.     

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.     

Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.     

MnSOD antisense treatment and exercise-induced protection against arrhythmias

Exercise provides protection against ischemia-reperfusion (I-R)-induced arrhythmias, myocardial stunning, and infarction. An exercise-induced increase in myocardial manganese superoxide dismutase (MnSOD) activity has been reported to be vital for protection against infarction. However, whether MnSOD is essential for exercise-induced protection against ventricular arrhythmias is unknown. We determined the effects of preventing the exercise-induced increase in MnSOD activity on arrhythmias during I-R resulting in myocardial stunning. Male rats remained sedentary or were subjected to successive bouts of endurance exercise. During in vivo myocardial I-R, the incidence of arrhythmias was significantly lower in the exercise-trained rats than in the sedentary rats as evidenced by the arrhythmia. When exercised rats were pretreated with antisense oligonucleotides directed against MnSOD, protection from arrhythmias was attenuated. Moreover, I-R resulted in significant increases in nitro-tyrosine (NT) in the sedentary group. Exercise abolished this I-R-induced NT formation but this protection was unchanged by antisense treatment. Protein carbonyls were increased by I-R, but neither exercise nor antisense treatment impacted carbonyl formation. These data demonstrate that an exercise-induced increase in MnSOD activity is important for protection against arrhythmias. The mechanism by which MnSOD provides protection does not appear to be linked to protein nitrosylation or oxidation.     

In vivo measurement of the dynamic 3-D kinematics of the ovine stifle joint

The ovine stifle joint is a promising animal model for investigation of joint mechanobiology. A method for in vivo measurement of dynamic 3-D kinematics of the ovine stifle joint is described (accuracy: 0.36 +/- 0.39 mm). Inter-subject variability in kinematics is greater than both intra-subject and inter-session variability. For future studies in which joint kinematics are measured prior to and following controlled orthopaedic interventions, pooling of data should be avoided and each subject should act as its own control.