Paramagnetic probes in NMR and EPR studies of membrane enzymes

The applications of paramagnetic probes to problems of structure and mechanism are discussed from the point of view of the membrane enzymologist. Problems unique to membrane systems are discussed, and a variety of nuclear and paramagnetic probes are evaluated. Three membrane ATPase (kidney (Na+ + K+)-ATPase, Ca2+-ATPase from sarcoplasmic reticulum and Mg2+-ATPase from kidney) are used to describe the types of experiments which can be done, the information which can be obtained and the limitations involved. Nuclear relaxation studies employing 1H, 7Li+, 31P and 205Tl+ nuclei are described. The advantages and disadvantages of Mn2+, Gd3+ and Cr3+ as paramagnetic probes are discussed in terms of the three ATPases. The theory and interpretation of Mn2+ and Gd3+ EPR spectra are evaluated in studies with the (Na+ + K+)-ATPase and Ca2+-ATPase, respectively.     

Magnesium deficiency enhances resistance to paraquat toxicity in bean leaves

Effects of deficient (20mmol m^?3) and sufficient (1000 mmol m^?3) magnesium (Mg) supply and of varied light intensity (100 μmol m^?2 s^?1 to 580 μmol m^?2 s^?1) on paraquat-dependent chlorophyll destruction in bean (Phaseolus vulgaris) plants grown in nutrient solution were studied over a 12-d period using leaf discs or intact primary leaves. Treatment of leaf discs with 10mmol m ³ paraquat for 15h caused severe chlorophyll loss, especially with increasing light intensity. This chlorophyll destruction by paraquat was very much higher in Mg-sufficient than Mg-deficient leaves. The occurrence of paraquat resistance in Mg deficient leaves was already apparent after 6d growth in nutrient solution, i.e. before any decrease in chlorophyll or growth by Mg deficiency was evident. Also, following foliar application of paraquat (10–140 mmol m^?3) to intact plants, Mg-deficient plants were much more resistant to paraquat, even following longer exposure duration (72 h) and four to 14 times higher paraquat concentrations than those received by Mg sufficient plants. From experiments where exogenous scavengers of superoxide radical (O₂^.-), hydroxyl radical (OH^·) and singlet oxygen (¹O₂) were applied to leaf discs, it appears that O₂^.-, and partly, OH^· are the main O₂ species which contribute to chlorophyll destruction by paraquat. The results demonstrate that Mg-deficient bean plants become highly resistant to O₂^.--mediated and light-induced paraquat injury. The mode of this paraquat resistance is attributed to well-known stimulative effects of Mg deficiency on O₂^.- and H₂O₂ scavenging enzymes and antioxidants.     

Alcohol dehydrogenase isozymes inGossypium hirsutum and its putative diploid progenitors: The biochemical consequences of enzyme multiplicity

Electrophoretic variation in alcohol dehydrogenase (ADH) was examined in tetraploidGossypium hirsutum and its putative diploid progenitorsG. ramondii, G. herbaceum, and a close relative,G. arboreum. All the diploids had three isozymes, while strains of the tetraploidG. hirsutum had either 4 or 6. Each isozyme was eluted from starch gels and significant differences in activity were noted between several of the isozymes relative to pH, substrate, temperature and salinity. This suggests that an increase in enzyme heterozygosity can result in higher levels of developmental homeostasis, but it depends on the isozyme alleles involved. Michigan Agricultural Experiment Station Journal Article No. 10379.     

Effect of food limitation on food consumption and reproductive allocation by adult milkweed bugs, Oncopeltus fasciatus

Male/female pairs of adult milkweed bugs, Oncopeltus faciatus were offered reduced food rations (100 mg milkweed seeds/week, 50 mg seeds/week, and 25 mg seeds/week) at 232C, 14L : 10D photoperiod. The high rate of food consumption normally occurring during the teneral period (first 8 days posteclosion) and subsequent food consumption were reduced on both reduced rations. Initial fresh weight was lost by bugs on both reduced rations and their weekly fresh body weight tended to remain less than bugs on the 100 mg seeds/week ration.Normal onset of egg production (about 3 weeks posteclosion) was delayed 6 weeks and normal length of a female's life (13–14 weeks) was increased 7–8 weeks on the 25 mg seeds/week ration. Rate of egg production exhibited a linear decrease with decrease in food ration on both reduced rations due to an increased interclutch interval, while fresh egg weight, duration of egg stage, number of eggs/clutch and percentage of clutches containing fertile eggs were little affected.Gross egg production efficiency (efficiency of conversion of ingested food into eggs = dry mg egg production × 100/dry mg food ingested) was high on both the 100 and 50 mg seeds/week rations (27.9% and 21.1%, respectively) and low (7.7%) on the 25 mg seeds/week ration. These data are discussed in the context of the reciprocal interaction between nutrient intake and egg production.     

Magnesium (and trace substance) deficiencies in the pathogenesis of cancer

Except for a few experimental models of magnesium (Mg)-deficiency-induced neoplasms, less attention has been paid in the past quarter century in the Western world to this macromineral than to the trace elements; e.g., selenium (Se) and zinc (Zn), and to vitamins, deficiencies of which are each considered probable factors in oncogenesis. Although early epidemiologic studies showed an inverse correlation between the amount of Mg in soil and water and the incidence of (gastric) cancer, and several animal studies supported the premise that Mg has a prophylactic effect against induction of cancer, other studies showed that Mg supplementation increased the growth of established experimental tumors. Thus, enthusiasm for this approach subsided. The early epidemiologic findings have since been confirmed, and there have been studies demonstrating the importance of Mg in maintaining immunocompetence, and others indicating that immunodeficiencies increase susceptibility to the development of cancer. Evidence has now accrued that indicates that Mg deficiency increases susceptibility to chemical oncogens. The abnormal metabolism of tryptophan (yielding a carcinogenic metabolite) that indicates functional or absolute pyridoxine deficiency is an indirect clue to Mg deficiency. Vitamin B₆-activated enzymes require Mg as a cofactor. However, the early warnings against the use of Mg as part of an antineoplastic program against established cancer were justified, since rapidly metabolizing cells (such as cancers) are dependent on Mg. There are similarities between experiences with Mg and with Se and Zn. All three are required for normal metabolism; Se also protects against free radicals in the environment. Mg and Zn have increased established tumor growth, and their depletion has been applied to antineoplastic programs, with risks comparable to those of using antimetabolic agents.     

Limitation of seedling growth by potassium and magnesium supply for two ectomycorrhizal tree species of a Central African rain forest and its implication for their recruitment

In the ectomycorrhizal caesalpiniaceous groves of southern Korup National Park, the dominant tree species, Microberlinia bisulcata, displays very poor in situ recruitment compared with its codominant, Tetraberlinia bifoliolata. The reported ex situ experiment tested whether availabilities of soil potassium and magnesium play a role. Seedlings of the two species received applications of K and Mg fertilizer in potted native soil in a local shade house, and their responses in terms of growth and nutrient concentrations were recorded over 2 years. Amended soil concentrations were also determined. Microberlinia responded strongly and positively in its growth to Mg, but less to K; Tetraberlinia responded weakly to both. Added Mg led to strongly increased Mg concentration for Microberlinia while added K changed that concentration only slightly; Tetraberlinia strongly increased its concentration of K with added K, but only somewhat its Mg concentration with added Mg. Additions of Mg and K had small but important antagonistic effects. Microberlinia is Mg‐demanding and apparently Mg‐limited in Korup soil; Tetraberlinia, whilst K‐demanding, appeared not to be K‐limited (for growth). Added K enhanced plant P concentrations of both species. Extra applied Mg may also be alleviating soil aluminum toxicity, and hence improving growth indirectly and especially to the benefit of Microberlinia. Mg appears to be essential for Microberlinia seedling growth and its low soil availability in grove soils at Korup may be an important contributing factor to its poor recruitment. Microberlinia is highly shade‐intolerant and strongly light‐responding, whilst Tetraberlinia is more shade‐tolerant and moderately light‐responding, which affords an interesting contrast with respect to their differing responses to Mg supply. The study revealed novel aspects of functional traits and likely niche‐partitioning among ectomycorrhizal caesalps in African rain forests. Identifying the direct and interacting indirect effects of essential elements on tropical tree seedling growth presents a considerable challenge due the complex nexus of causes involved.     

A mechanism coordinating root elongation,endodermal differentiation,redox homeostasis and stress response

Root growth relies on both cell division and cell elongation, which occur in the meristem and elongation zones, respectively. SCARECROW (SCR) and SHORT-ROOT (SHR) are GRAS family genes essential for root growth and radial patterning in the Arabidopsis root. Previous studies showed that SCR and SHR promote root growth by suppressing cytokinin response in the meristem, but there is evidence that SCR expressed beyond the meristem is also required for root growth. Here we report a previously unknown role for SCR in promoting cell elongation. Consistent with this, we found that the scr mutant accumulated a higher level of reactive oxygen species (ROS) in the elongation zone, which is probably due to decreased expression of peroxidase gene 3, which consumes hydrogen peroxide in a reaction leading to Casparian strip formation. When the oxidative stress response was blocked in the scr mutant by mutation in ABSCISIC ACID 2 (ABA2) or when the redox status was ameliorated by the upbeat 1 (upb1) mutant, the root became significantly longer, with longer cells and a larger and more mitotically active meristem. Remarkably, however, the stem cell and radial patterning defects in the double mutants still persisted. Since ROS and peroxidases are essential for endodermal differentiation, these results suggest that SCR plays a role in coordinating cell elongation, endodermal differentiation, redox homeostasis and oxidative stress response in the root. We also provide evidence that this role of SCR is independent of SHR, even though they function similarly in other aspects of root growth and development.     

Eimeria tenella: Incomplete Excystation in the Presence of EDTA in a Taurodeoxycholate-Based Medium

ABSTRACT. Normally, sporozoites of Eimeria tenella are efficiently excysted in vitro with trypsin and bile salts. However, a one hour treatment at °40C with a chelator-supplemented excystation medium (purified trypsin and chymotrypsin, taurodeoxycholate and ethylenediaminetetraacetic acid in buffered saline) produced incomplete excystation. The treatment removed the sporocyst plug and left an opened sporocyst containing motile sporozoites, but the release of sporozoites was greatly reduced (<12% release). Some of the sporozoites extended a portion of their anterior end through the sporocyst opening then retracted it into the sporocyst. Sporozoites were released when magnesium was added to the chelator-supplemented medium. Manganese was less effective and calcium was ineffective in producing release. Also, sporozoites were released when the incompletely excysted sporocysts were transferred to buffered saline with albumin and this became the basis for a new assay. The assay demonstrated that ethylenediaminetetraacetic acid reduced release in the presence of taurodeoxycholate but not in its absence. Hydrophobic and hydrophilic chelators were tested in the assay. Ethylene-dioxy diethylene-dinitrilotetraacetic acid and 8-hydroxyquinoline were inactive. The chelator 1,10-phenanthroline did not require bile salt to reduce release. The inhibitory effects by phenanthroline were eliminated in the presence of magnesium or manganese, while calcium had no effect. Thus, although certain chelators can inhibit release, a consistent correlation between chelation and inhibition of release has not been established. The application of ethylenediaminetetraacetic acid with taurodeoxycholate as a reversible inhibitor of release is discussed.     

The induction of autophagy by mechanical stress

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.