Photosynthetic performance of invasive Pinus ponderosa and Juniperus virginiana seedlings under gradual soil water depletion.

Changes in climate, land management and fire regime have contributed to woody species expansion into grasslands and savannas worldwide. In the USA, Pinus ponderosa P.&C. Lawson and Juniperus virginiana L. are expanding into semiarid grasslands of Nebraska and other regions of the Great Plains. We examined P. ponderosa and J. virginiana seedling response to soil water content, one of the most important limiting factors in semiarid grasslands, to provide insight into their success in the region. Photosynthesis, stomatal conductance, maximum photochemical efficiency of PSII, maximum carboxylation velocity, maximum rate of electron transport, stomatal limitation to photosynthesis, water potential, root-to-shoot ratio, and needle nitrogen content were followed under gradual soil water depletion for 40 days. J. virginiana maintained lower L(s), higher A, g(s), and initial F(v)/F(m), and displayed a more gradual decline in V(cmax) and J(max) with increasing water deficit compared to P. ponderosa. J. virginiana also invested more in roots relative to shoots compared to P. ponderosa. F(v)/F(m) showed high PSII resistance to dehydration in both species. Photoinhibition was observed at approximately 30% of field capacity. Soil water content was a better predictor of A and g(s) than Psi, indicating that there are other growth factors controlling physiological processes under increased water stress. The two species followed different strategies to succeed in semiarid grasslands. P. ponderosa seedlings behaved like a drought-avoidant species with strong stomatal control, while J. virginiana was more of a drought-tolerant species, maintaining physiological activity at lower soil water content. Differences between the studied species and the ecological implications are discussed.

Ecology of Increasing Diseases: Population Growth and Environmental Degradation.

The World Health Organization (WHO) and other organizations report that the prevalence of human diseases during the past decade is rapidly increasing. Population growth and the pollution of water, air, and soil are contributing to the increasing number of human diseases worldwide. Currently an estimated 40% of world deaths are due to environmental degradation. The ecology of increasing diseases has complex factors of environmental degradation, population growth, and the current malnutrition of about 3.7 billion people in the world.

Calcium-calmodulin-stimulated phosphorylation of rat parotid secretion granule proteins.

In studies designed to determine the mechanism by which Ca++ and calmodulin stimulate the fusion of parotid secretion granules with plasma membrane vesicles, the hypothesis tested was that Ca++ and calmodulin act by stimulating protein phosphorylation. It was earlier found that Ca++ and calmodulin, but neither alone, stimulated the phosphorylation of four secretion granule proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the degree of phosphorylation of a 36-kDa protein. Further studies have shown that in the presence of an optimal concentration of calmodulin (2.4 microM), half-maximal activation of phosphorylation of the four proteins occurred at approx. 8 microM Ca++, and at a maximally effective Ca++ concentration (10(-4) M), half-maximal stimulation occurred at calmodulin concentrations between 0.13 and 1.1 microM for the different proteins. The studies now described also demonstrate that the need for calmodulin for stimulating the phosphorylation, but not the dephosphorylation, is specific; two other Ca(++)-binding proteins, parvalbumin and troponin, could not replace calmodulin in stimulating phosphorylation of the four secretion granule proteins, but either one could substitute for calmodulin in stimulating dephosphorylation of the 36-kDa protein. Additionally, the phosphorylated proteins appear to be located on the granule surface. When secretion granules were subjected to mild treatment with a concentration of trypsin that did not lyse the granules, the 31-, 36-, 55-, 58- and 64-kDa proteins were no longer observed. In the presence of optimal concentrations of Ca++ and calmodulin, a dose-dependent inhibition of the phosphorylation of the various proteins by two calmodulin antagonists, trifluoperazine and calmidazolium, was observed; 50% inhibition of phosphorylation of the different proteins was obtained at approx. 20-40 microM trifluoperazine and at about 2.5-3.0 microM calmidazolium. Inhibition of the dephosphorylation of the 36-kDa protein required greater concentrations of trifluoperazine and calmidazolium; 128 microM and 50 microM, respectively. These results are consistent with the hypothesis that the phosphorylation of one or more of the 31-, 55-, 58- and 64-kDa proteins, but not the dephosphorylation of the 36-kDa protein, may be involved in the action of Ca++ and calmodulin in secretion granule-plasma membrane fusion.

Effects of calcium and calmodulin on the binding of rat parotid secretion granules to the plasma membrane.

Since numerous studies suggest that Ca++ and calmodulin may modulate the fusion of secretion granules to the plasma membrane which takes place in exocytosis, we have examined the role of calcium and calmodulin in the binding of isolated parotid secretion granules to plasma membrane vesicles. 125I-labeled inside-out plasma membrane vesicles were incubated with secretion granules, the mixture was layered over 20% sucrose, the gradient was centrifuged, and the amount of 125I in the granule pellet was determined. Addition of Ca++ (20 nM to 10 microM) produced a concentration-dependent increase in the binding of 125I-labeled plasma membrane vesicles to the secretion granules, reaching a maximum value at 10 microM free Ca++; half-maximal binding occurred at 400 nM. Neither right-side-out parotid plasma membrane vesicles nor inside-out pancreatic islet plasma membrane vesicles bound to granules in the presence of 1 microM Ca++. Calmodulin produced a concentration-dependent increase in binding above that of Ca++ alone, and this effect was inhibited by the calmodulin antagonists, trifluoperazine and calmidazolium. Incubation of secretion granules with octadecylrhodamine B (R18)-loaded inside-out plasma membrane vesicles and 2 microM Ca++ caused de-quenching of fluorescence, indicating that the lipids in the granule membrane and the plasma membrane had intermixed. Added calmodulin increased the fluorescence two-fold above that with Ca++ alone. These results suggest that Ca++ and calmodulin may play a role in parotid gland exocytosis by modulating the interaction between the secretion granules and plasma membrane.

Ca(++)-calmodulin-dependent phosphorylation and dephosphorylation of rat parotid secretion granules.

In our studies on the control of fusion of secretion granules with the plasma membrane which occurs during exocytosis, we have recently found that Ca++ and calmodulin stimulated the fusion of isolated parotid secretion granules with isolated inside-out plasma membrane vesicles. We are now examining the possibility that they do so by stimulating protein phosphorylation. Secretion granules were isolated from rat parotid by differential and gradient centrifugation and incubated with [Y32P]ATP. The granules were solubilized with sodium dodecylsulfate and the proteins resolved on a 7-15% polyacrylamide gel. Calmodulin plus Ca++, but neither alone, stimulated phosphorylation of four proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the phosphorylation of a 36 kDa protein. Trifluoperazine and calmidazolium inhibited these effects. The results suggest that Ca++ and calmodulin may facilitate fusion of secretion granules with the plasma membrane by changing the phosphorylation state of one or more secretion granule proteins.

Effect of sulfhydryl reagents on pancreatic islet secretion granule-plasma membrane interaction.

To determine what role, if any, sulfhydryl groups may play in the fusion of islet secretion granules with the plasma membrane that takes place during exocytosis, we have studied the effect of several sulfhydryl-binding reagents, reducing agents, and oxidizing agents on the binding of 125I-labeled inside-out plasma membrane vesicles to isolated secretion granules. Three sulfhydryl-binding reagents, p-hydroxymercuribenzoate, Hg++, and N-ethyl maleimide, stimulated this binding, and the stimulation was greater in the absence of Ca++ than in its presence. In contrast, the three reducing agents used, glutathione, cysteine, and sodium bisulfite, inhibited the binding. Of the oxidizing agents, oxidized glutathione inhibited binding, whereas menadione and o-iodosobenzoate stimulated. The actions of Hg++ and glutathione were found to be on the secretion granules rather than the plasma membrane vesicles. It is concluded that the presence of a preponderance of sulfhydryl groups on the secretion granule membranes tends to limit their interaction with the plasma membrane and that these must be removed or masked for maximum fusion to occur.

Synthesis of glycosaminoglycans by islets of Langerhans.

Incorporation of (35S)-sulfate into glycosaminoglycans (GAG) of toadfish islets of Langerhans in vitro was examined. (35S)-sulfated GAG were synthesized by a component of the microsomal fraction, and subsequently transferred to the secretion granules, mitochondria and nuclei. The predominant type of GAG synthesized was heparan sulfate, but chondroitin 4- and 6-sulfate and dermatan sulfate were also found.

Role of calcium and calmodulin in the interaction between islet cell secretion granules and plasma membranes.

The effects of calcium and calmodulin on the binding of islet secretion granules to plasma membrane vesicles was studied. Free Ca++ (10(-7) to 10(-4) M) produced a concentration-dependent increase in the binding of inside-out. 125I-labeled plasma membrane vesicles to secretion granules; maximum stimulation occurred at 10(-5) M. By contrast, Ca++ had no effect on binding when either inside-out red blood cell or right-side-out islet plasma membrane vesicles were used. Calmodulin (1 microM), in the presence of 10(-4) M Ca++, increased the binding two-fold as compared to Ca++ alone, and trifluoperazine (TFP), and inhibitor of calmodulin, produced a concentration-dependent inhibition of the calmodulin effect. TFP also inhibited the binding produced by 10(-4) M Ca++ in the absence of exogenous calmodulin. These results suggest that Ca++ in the presence of calmodulin modulates the interaction between islet secretion granules and the cytoplasmic surface of the plasma membrane.

Effect of sulfhydryl-binding reagents on release of somatostatin immunoreactivity from secretion granules.

The effect of various sulfhydryl-binding reagents on the release of somatostatin immunoreactivity (SRIF-I) from secretion granules of the islets of Langerhans was studied. The granules were isolated from toadfish islet tissue by differential centrifugation in sucrose. They were then incubated in the test medium, pelleted, and the supernatant was assayed for SRIF-I using a radioimmunoassay. When the granules were incubated in isotonic saline, about 20% of the total SRIF-I in the granules was released into the medium. Three mercurials (p-hydroxymercuribenzoate, phenylmercuric acetate and Hg++) and an arsenical (diphenylchloroarsine) increased SRIF-I release 1.5 to 2-fold. In contrast, other heavy metals (Co++ and Cd++), other arsenicals (dichlorophenylarsine and AsO-2), alkylating agents (N-ethylmaleimide and iodoacetic acid), an oxidizing agent (oxidized glutathione), a reducing agent (reduced glutathione), and an amino group-binding reagent (trinitrobenzene sulfonic acid), had no effect. These results suggest that sulfhydryl groups may be present on the somatostatin secretion granules and, if so, that they differ from those previously demonstrated on the insulin- and glucagon-secretion granules.

Uptake of amino acids by islet of Langerhans and other tissues of the toadfish.

The uptake of injected radioactive amino acids by the isolated islet and other tissues of the toadfish has been examined. The islet and most other tissues appear to actively transport most amino acids and, of the tissues studied, the islet is one of the most active in this regard. Most tissues accumulated the dicarboxylic acids to the greatest extent, with the basic amino acids and the neutral amino acids with unbranched side chains also being taken up well. However, each tissue showed a unique pattern of uptake of the different amino acids, and islet was distinguished by its high uptake of glycine 15 min after injection. The pattern of uptake of amino acids by toadfish islet does not appear to be related to their relative ability to stimulate insulin release from islets of other species or to their relative rates of metabolism, but does correlate well with the content of free amino acids in mouse islets and with the sum of the amino acid contents of anglerfish proinsulin and proglucagon.

Effect of alloxan on islet tissue permeability: protection and reversal by dithiols.

The ability of dithiol compounds with different spacing between the thiol (SH) groups to protect against and reversed the action of alloxan on islet tissue permeability, and their ability to inhibit the reaction between alloxan and glutathione (which results in the formation of a compound with a 305 nm absorption maximum) have been examined. Treatment of toadfish islet slices with alloxan markedly increased their permeability to D-mannitol-1-14C, which normally is restricted to the extracellular space. Pretreatment of the slices with 2,3-dimercaptopropanol (BAL) or 1,4-dimercaptobutane (DMB) before treatment with alloxan completely protected them against this action of alloxan, whereas 1,5-dimercaptopentane (DMP) and 1,6-dimercaptohexane (DMH) partially protected and 1,8-dimercapto-octane (DMO) had no effect. When islets were first treated with alloxan and then treated with the dithiols, BAL and DMB reverse the action of alloxan to the greatest extent, DMP was less effective, and DMH had no effect. The effect of the dithiols on the reaction between alloxan and glutathione was consistent with their effects on the alloxan-induced increase in islet permeability; BAL and DMB were the strongest inhibitors, DMP and DMH inhibited to a lesser degree and DMO did not inhibit. These studies support the hypothesis that alloxan damages islet cell membranes by reacting with membrane dithiols formed by two SH groups which are relatively close together.