No effect of menstrual cycle phase on glucose kinetics and fuel oxidation during moderate-intensity exercise

Resting and exercise fuel metabolism was assessed in three different phases of the menstrual cycle, characterized by different levels of estrogen relative to progesterone: early follicular (EF, low estrogen and progesterone), midfollicular (MF, elevated estrogen, low progesterone), and midluteal (ML, elevated estrogen and progesterone). It was hypothesized that exercise glucose utilization and whole body carbohydrate oxidation would decrease sequentially from the EF to the MF to the ML phase. Normal-weight healthy females, experiencing a regular menstrual cycle, were recruited. Subjects were moderately active but not highly trained. Testing occurred after 3 days of diet control and after an overnight fast (12-13 h). Resting (2 h) and exercise (50% maximal O(2) uptake, 90 min) measurements of whole body substrate oxidation, tracer-determined glucose flux, and substrate and hormone concentrations were made. No significant difference was observed in whole body fuel oxidation during exercise in the three phases (nonprotein respiratory exchange ratio: EF 0.84 +/- 0.01, MF 0.85 +/- 0.01, ML 0.85 +/- 0.01) or in rates of glucose appearance or disappearance. There were, however, significantly higher glucose (P < 0.05) and insulin (P < 0.001) concentrations during the first 45 min of exercise in the ML phase vs. EF and MF phases. In conclusion, whole body substrate oxidation and glucose utilization did not vary significantly across the menstrual cycle in moderately active women, either at rest or during 90 min of moderate-intensity exercise. During the ML phase, however, this similar pattern of substrate utilization was associated with greater glucose and insulin concentrations. Both estrogen and progesterone are elevated during the ML phase of the menstrual cycle, suggesting that one or both of these sex steroids may play a role in this response.     

Postprandial leg uptake of triglyceride is greater in women than in men

The postprandial excursion of plasma triglyceride (TG) concentration is greater in men than in women. In this study, the disposition of dietary fat was examined in lean healthy men and women (n = 8/group) in either the overnight-fasted or fed (4.5 h after breakfast) states. A [14C]oleate tracer was incorporated into a test meal, providing 30% of total daily energy requirements. After ingestion of the test meal, measures of arteriovenous differences in TG and 14C across the leg were combined with needle biopsies of skeletal muscle and adipose tissue and respiratory gas collections to define the role of skeletal muscle in the clearance of dietary fat. The postprandial plasma TG and 14C tracer excursions were lower (P = 0.04) in women than in men in the overnight-fasted and fed states. Women, however, had significantly greater limb uptake of total TG compared with men on both the fasted (3,849 +/- 846 vs. 528 +/- 221 total micro mol over 6 h) and fed (4,847 +/- 979 vs. 1,571 +/- 334 total micromol over 6 h) days. This was also true for meal-derived 14C lipid uptake. 14C content of skeletal muscle tissue (micro Ci/g tissue) was significantly greater in women than in men 6 h after ingestion of the test meal. In contrast, 14C content of adipose tissue was not significantly different between men and women at 6 h. The main effect of nutritional state, fed vs. fasted, was to increase the postmeal glucose (P = 0.01) excursion (increase from baseline) and decrease the postmeal TG excursion (P = 0.02). These results support the notion that enhanced skeletal muscle clearance of lipoprotein TG in women contributes to their reduced postprandial TG excursion. Questions remain as to the mechanisms causing these sex-based differences in skeletal muscle TG uptake and metabolism. Furthermore, nutritional state can significantly impact postprandial metabolism in both men and women.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Biochemical evidence for multiple dimeric states of the Sinorhizobium meliloti DctD receiver domain

X-ray crystal structures suggest very different dimeric states for the inactive and active forms of the two-component receiver domain of Sinorhizobium meliloti DctD, a sigma(54)-dependent AAA+ ATPase. Moreover, the receiver domain in crystals grown from unphosphorylated protein is refractory to phosphorylation whereas solution protein is fully phosphorylatable, and equilibrium analytical ultracentrifugation data are consistent with solution dimers for both phosphorylated and unphosphorylated forms of the protein. Here we report biochemical data consistent with the presence of multiple dimeric conformations in the inactive and active states, and evidence for significant change in the dimeric state upon activation by phosphorylation or binding of Mg(2+) and BeF(3)(-).     

Slow heating of barley aleurone layers to heat-shock temperature preserves heat-shock-sensitive cellular properties

In barley (Hordeum vulgare L. cv. Himalaya) aleurone layers, heat shock causes the selective suppression of α-amylase synthesis by destabilizing this secretory protein's mRNA. The lamellar stacks of the endoplasmic reticulum (ER), which serve as the site of α-amylase mRNA translation, are dissociated by heat shock, suggesting that heat-shock-induced changes in ER may be important in selectively targeting α-amylase mRNAs for destabilization. We have found that samples maintained at heat-shock temperature (40°C) for 18 h recover the ability to synthesize α-amylase and that the ER membranes in these samples contain membrane phospholipids with enhanced levels of fatty acid saturation. This present study investigated whether gradual warming to 40°C over 3-6 h (ramping) would preserve α-amylase synthesis by permitting ER membrane phospholipid retailoring during the gradual temperature increase. Analyses by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed that α-amylase synthesis was markedly increased in ramped samples. Furthermore, northern hybridization analyses and transmission electron microscopy showed that these samples had increased α-amylase mRNA levels and stacks of ER lamellae, respectively. Gas chromatographic analyses of ER membrane phospholipids indicated that the fatty acids of ramped samples were more saturated than their heat-shocked counterparts. These data indicate that heat-induced increases in aleurone ER membrane phospholipid fatty acid saturation may be important in maintaining secretory protein expression at normally nonpermissive heat-shock temperatures.     

Amiloride is an ineffective conditioned stimulus in taste aversion learning in C57BL/6J and DBA/2J mice

The epithelial sodium channel (ENaC) blocker amiloride has been shown to increase the behaviorally measured NaCl detection threshold in mice. In this study, a conditioned taste aversion (CTA) paradigm was used to examine whether 100 microM amiloride has a perceptible taste that could contribute to this observed decrease in behavioral responsiveness. Eighty-four C57BL/6J (B6) and 64 DBA/2J (D2) mice were divided into eight groups (n=8-12 per group), in which half received an injection of 0.15 M LiCl (2 mEq/kg) and the other half an equivalent saline injection, in three conditioning trials. The four conditioned stimuli were 100 microM amiloride hydrochloride, water, 0.1 and 0.3 M NaCl. Neither strain demonstrated acquisition of a CTA to amiloride in a brief-access (BA) taste test (5 s trials in the gustometer). Although 0.3 M NaCl is inherently aversive, its pairing with LiCl led to significantly further decreases in licking during the BA test on salt trials in both strains. The D2 strain clearly avoided 0.1 M NaCl, whereas avoidance of this stimulus was more equivocal in B6 mice. The inefficacy of amiloride to serve as a conditioned stimulus in taste aversion learning involving three LiCl pairings suggests that the effects of this ENaC blocker on taste-related behavioral responses to NaCl are likely due to its pharmacological interference with sodium taste transduction.     

Use of immunohistochemistry to diagnose chytridiomycosis in dyeing poison dart frogs (Dendrobates tinctorius)

Chytridiomycosis, caused by Batrachochytrium dendrobatidis, is an emerging disease of both wild and captive amphibians, posing a threat to their survival in many parts of the world. As the disease can be difficult to diagnose on routine pathologic sections, the purpose of this study was to develop an additional method for visualization. To accomplish this, immunohistochemical staining was applied to histologic skin sections from four experimentally infected Dyeing poison dart frogs (Dendrobates tinctorius). Staining of the positive tissue sections was distinct and readily visualized, making this technique a valuable ancillary diagnostic test for this important disease.     

Tome-1, a trigger of mitotic entry,is degraded during G1 via the APC

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.     

Effects of mesozooplankton removal and ammonium addition on planktonic trophic structure during a bloom of the Texas 'brown tide': a mesocosm study

A bloom of the alga Aureoumbra lagunensis, known as the Texas‘brown tide’, persisted in the Laguna Madre of Texasfor most of the 1990s. The dominant mesozooplankter in LagunaMadre, Acartia tonsa, does not feed on A. lagunensis, and duringblooms there are few other suitably sized phytoplankton cellsavailable to feed on. We hypothesized that these copepods increasedtheir feeding on microzooplankton, thereby reducing grazingpressure by microzooplankton on A. lagunensis and contributingto the persistence of this bloom. A mesocosm experiment wascarried out to test this hypothesis during the summer of 1999.Twelve fiberglass corral-type mesocosms were deployed in thefield for 16 days, each enclosing 1.2 m³ of Laguna Madre waterand 1.1 m² of natural benthos. Mesozooplankton were removedfrom six mesocosms with a 153 µm mesh dip net every 4days; the other six mesocosms were treated in the same way,except that the contents of the net were returned to the mesocosm.For each zooplankton treatment, half of the mesocosms were dosedwith 40 µm NH₄ at 4 day intervals, and half received noadditions. Phytoplankton populations in these mesocosms at thestart of the experiment were dominated by A. lagunensis andthe cyanobacterium Synechococcus spp. Growth rates of A. lagunensiswere higher in mesocosms without ammonium additions, providingno evidence for nitrogen limitation. Acartia tonsa populationswere reduced by 50% in the zooplankton removal mesocosms, andciliate populations were significantly higher. The increasein ciliate population had no significant impact on A. lagunensispopulation dynamics, however, providing no evidence to supportthe hypothesis that a trophic cascade reducing microzooplanktonpopulations contributed to the persistence of the brown-tidebloom. In contrast, populations of Synechococcus sp. showedevidence of both ‘top–down’ and ‘bottom–up’control; they grew faster in nutrient addition mesocosms andhad lower populations in mesocosms with increased densitiesof ciliate grazers.