Evidence for a novel bursting mechanism in rodent trigeminal neurons.

We investigated bursting behavior in rodent trigeminal neurons. The essential mechanisms operating in the biological systems were determined based on testable predictions of mathematical models. Bursting activity in trigeminal motoneurons is consistent with a traditional mechanism employing a region of negative slope resistance in the steady-state current-voltage relationship (Smith, T. G. 1975. Nature. 253:450-452). However, the bursting dynamics of trigeminal interneurons is inconsistent with the traditional mechanisms, and is far more effectively explained by a new model of bursting that exploits the unique stability properties associated with spike threshold (Baer, S. M., T. Erneux, and J. Rinzel. 1989. SIAM J. Appl. Math. 49:55-71).     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

A MADS domain gene involved in the transition to flowering in Arabidopsis

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUS LIKE 20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long-day and constitutive pathways of floral induction the expression of AGL20 is down-regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over-expression of the orthologous gene from mustard, MADSA, in the classical short-day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.     

Acute hibernation decreases myocardial pyruvate carboxylation and citrate release

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.     

Bovine spermatozoal head size variation and evaluation of a separation technique based on this size

The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency.     

Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.     

The conformational states of Mg·ATP in water

The conformational equilibria of Mg·ATP in solution is studied using molecular dynamics (MD) augmented with umbrella sampling methods. Free energy comparisons show that the Mg²⁺ ion is equally likely to coordinate the oxygens of the two end phosphates, or of all three phosphates. The MD trajectories reveal two major degrees of freedom of the Mg·ATP molecule in solution, and we compute the free energy as a function of these variables, and determine its elastic properties. Comparing the free energy function with several crystallographic structures of ATP analogs, we find that the crystal structures correspond to states where ATP would be elastically strained. The average water density around Mg·ATP is investigated to show the average number of hydrogen bonds and the hydrophobicity.