Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. I. Experimental measures.

In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a "two-step" low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the "equilibrium" (infinitely slow) cooling behavior and the osmotically inactive cell volume (V(b)) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5 degrees C/min. Stereological analysis of the slam frozen (>1000 degrees C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DeltaX = 64 microm; original sinusoid (vascular) radius, r(vo) = 18.4 microm; and length of the Krogh cylinder, L = 0.71 microm (based on an isolated frog hepatocyte cell diameter of 16 microm). A significant observation was that approximately 24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r(2) = 28.7 microm, was defined (in addition to the original sinusoid radius, r(vo) = 18.4 microm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle-van't Hoff plot, the osmotically inactive cell volume, V(b) was obtained as 0.4. V(o) (where V(o) is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min.     

Nitrogen processing in the hyporheic zone of a pastoral stream

The distribution of nitrogen-transforming processes, and factors controlling their rates, were determined within the hyporheic zone of a lowland stream draining agricultural land. In the field, physicochemical parameters were measured along a 10m-long hyporheic flow line between downwelling and upwelling zones. Sediment cores were retrieved from the stream bed surface, and from 20, 40 and 60cm deep in each zone, and in the laboratory, water from the corresponding depth was percolated through each core at the natural flow rate. Concentrations of nitrogen species and oxygen were measured before and after flow through each core. Denitrification was measured using a ¹⁵N-nitrate tracer. Shallow and downwelling zone samples were clearly distinct from deeper and upwelling zone samples in terms of physicochemical conditions, microbial processes and factors controlling nitrogen processing. Denitrification was highest in surface and downwelling zone cores, despite high oxygen levels, probably due to high pore-water nitrate concentrations in these cores and isolation of the denitrifying bacteria from oxygen in the bulk water by the hyporheic biofilms. Denitrification was limited by oxygen inhibition in the downwelling group, and by nitrate availability in the upwelling group. Strong evidence indicated that dissimilatory nitrate reduction to ammonium, occurred in almost all cores, and outcompeted denitrification for nitrate. In contrast, nitrification was undetectable in all but two cores, probably because of intense competition for oxygen. Field patterns and lab experiments indicated that the hyporheic zone at this moderately N-rich site is a strong sink for nitrate, fitting current theories that predict where hyporheic zones are nitrate sinks or nitrate sources.     

Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.     

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Sterol carrier protein-2 (SCP-2) and SCP-x are ubiquitous proteins found in all mammalian tissues. Although both proteins interact with fatty acids, their relative contributions to the uptake, oxidation, and esterification of straight-chain (palmitic) and branched-chain (phytanic) fatty acids in living cells has not been resolved. Therefore, the effects of each gene product on fatty acid metabolism was individually examined. Based on the following, SCP-2 and SCP-x did not enhance the uptake/translocation of fatty acids across the plasma membrane into the cell: i) a 2-fold increase in phytanic and palmitic acid uptake was observed at long incubation times in SCP-2- and SCP-x-expressing cells, but no differences were observed at initial time points; ii) uptake of 2-bromo-palmitate, a nonoxidizable, poorly metabolizable fatty acid analog, was unaffected by SCP-2 or SCP-x overexpression; and iii) SCP-2 and SCP-x expression did not increase targeting of radiolabeled phytanic and palmitic acid to the unesterified fatty acid pool. Moreover, SCP-2 and SCP-x expression enhanced fatty acid uptake by stimulating the intracellular metabolism via fatty acid oxidation and esterification. In summary, these data showed for the first time that SCP-2 and SCP-x stimulate oxidation and esterification of branched-chain as well as straight-chain fatty acids in intact cells.     

Ca-ATPase activity and protein composition of sarcoplasmic reticulum membranes isolated from skeletal muscles of typical hibernator,the ground squirrel Spermophilus undulatus

Ca-ATPase activity in sarcoplasmic reticulum (SR) membranes isolated from skeletal muscles of the typical hibernator, the ground squirrel Spermophilus undulatus, is about 2-fold lower than that in SR membranes of rats and rabbits and is further decreased 2-fold during hibernation. The use of carbocyanine anionic dye Stains-All has revealed that Ca-binding proteins of SR membranes, histidine-rich Ca-binding protein and sarcalumenin, in ground squirrel, rat, and rabbit SR have different electrophoretic mobility corresponding to apparent molecular masses 165, 155, and 170 kDa and 130, 145, and 160 kDa, respectively; the electrophoretic mobility of calsequestrin (63 kDa) is the same in all preparations. The content of these Ca-binding proteins in SR membranes of the ground squirrels is decreased 3–4 fold and the content of 55, 30, and 22 kDa proteins is significantly increased during hibernation.     

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.