Alfalfa rapidly remediates excess inorganic nitrogen at a fertilizer spill site.

By 19%, standard remediation techniques had significantly reduced the concentration of nitrate nitrogen (NO3- -N) in local ground water at the site of a 1989 anhydrous ammonia spill, but NO3- -N concentrations in portions of the site still exceeded the public drinking water standard. Our objective was to determine whether local soil and ground water quality could be improved with alfalfa (Medicago sativa L.). A 3-yr study was conducted in replicated plots (24 by 30 m) located hydrologically upgradient of the ground water under the spill site. Three alfalfa entries ['Agate', Ineffective Agate (a non-N2-fixing elite germplasm similar to Agate), and MWNC-4 (an experimental germplasm)] were seeded in the spring of 1996. Corn (Zea mays L.) or wheat (Triticum aestivum L.) was seeded adjacent to the alfalfa each year. Crops were irrigated with N-containing ground water to meet water demand. During the 3-yr period, about 540 kg of inorganic N was removed from the aquifer through irrigation of 4.9 million L water. Cumulative N removal from the site over 3 yr was 972 kg N ha(-1) in Ineffective Agate alfalfa hay, compared with 287 kg N ha(-1) for the annual cereal grain. Soil solution NO3- concentrations were reduced to low and stable levels by alfalfa, but were more variable under the annual crops. Ground water quality improved, as evidenced by irrigation water N concentration. We do not know how much N was removed by the N2-fixing alfalfas, but it appears that either fixing or non-N2-fixing alfalfa will effectively remove inorganic N from N-affected sites.

Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

Philanthropic endowments in general internal medicine.

We performed two surveys to uncover the status of philanthropic endowments in general internal medicine divisions. The initial survey of U.S. medical school departments of medicine found that only 14.1% of general internal medicine divisions hold endowments versus 21.9% of all other divisions, and that endowment sources for general medicine are atypical. The second survey of successfully endowed divisions found that sympathetic administrators and active pursuit of endowments were associated with endowment success. Aggressive pursuit of endowments, publicizing successes of general medicine, and consideration of endowment sources noted in this study are recommended to improve philanthropic contributions to general internal medicine.

Lumbar spinal stenosis in an elderly patient.

BACKGROUND: The general population is aging, and lumbar stenosis is one of the more frequent conditions observed in an orthopedic or neurosurgical practice. METHODS: This case presentation is of an 86-year-old male who developed lumbar spinal stenosis with a progressive neurologic deficit that caused severe leg pain, affected bladder function, and affected gait. Relevant medical literature is reviewed. RESULTS: Bladder function and gait returned after spinal surgery, and this patient's pain was greatly reduced. A multidisciplinary team applied therapy after surgery. The medical literature does not concentrate solely upon patients older than 80, but a few are included in studies of younger patients. CONCLUSIONS: This case report illustrates that a patient over 80 can have a successful outcome with multidisciplinary medical coverage of medical, surgical, rehabilitative, social, and psychological areas. More studies need to be done of these patients.

Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture.

1. Human cells (HeLa) were cultured for periods up to 48 h in growth medium in the absence or presence of a range of concentrations of cardiac glycosides. In some experiments the potassium concentration of the medium was varied between 0.3 mM and the usual 5 mM. 2. For periods up to 2 h in ouabain the association and dissociation rate constants were measured and the equilibrium binding constant (KD) calculated; the apparent equilibrium binding constant (K'D) was measured after 1-2 days growth in ouabain. 3. Ouabain had a K'D after 2 days of 2-6 nM in 5 mM K+ growth medium, a 4 fold greater blocking effect on sodium pumps after 2 days than expected from the association and dissociation rate constants measured in untreated or previously ouabain-treated cells. 4. This effect was: (a) approximately the same over a range of external potassium concentrations from 0.3 to 5 mM, although the absolute effect of ouabain over this range of potassium was much different; (b) probably not due to different isoforms of pumps in cells grown in ouabain compared to untreated cells; (c) apparently not a consequence of internalisation of pump-glycoside complexes. 5. We conclude that ouabain has only a limited access to sodium pumps in whole cells; this could be because sodium pumps cycle continuously through an inaccessible region of the plasma membrane. This effect needs to be considered both in the assessment of the magnitude of the long term effects of cardiac glycosides on cells, and in the measurement of the glycoside affinities of various isoforms of the pump.

The effects of monensin on the abundance of mRNA(alpha) and of sodium pumps in human cultured cells.

HeLa or MRC5-VI cells were grown for up to 1 day in media containing monensin at concentrations up to 10 microM. We measured the sodium pump density of the plasma membrane with [3H]ouabain and the mRNA for the alpha-subunit of the pump by hybridization to a cDNA probe. The sodium and potassium concentrations were measured under similar conditions, and in some experiments the rate of internalization of the sodium pumps estimated by using [3H]ouabain uptake into the cell. We found that the relationship between sodium pump density and [Na+]i was well described by Michaelis-Menten kinetics with a Km of 12 mM-[Na+]i and a Vmax twice the normal value. There was no obvious relationship between cell potassium and pump density. The relationship between sodium pump density and [Na+]i was the same as that found by growing the cells in low-potassium medium, so we conclude that the manner of raising [Na+]i is not important, merely the final value. In conditions of raised intracellular sodium there was an increase in the mRNA for the alpha-subunit of the pump, but there was no slowing of the rate of internalization of the pumps from the plasma membrane. We conclude that the increased density of pumps is due to an increased synthesis rather than a decreased internalization rate of pumps, suggesting that the cell can control the method of upregulation.

Transient changes in permeability in HeLa and L cells during detachment from a substrate.

The Na+ and K+ gradients of HeLa cells approach that of the medium during removal from a substrate with trypsin, but then recover during the next 15 min. The recovery is blocked by ouabain or the cold, but is unaffected by bumetanide. The effect is also obtained in cells which have no intercellular connexions, and in cells whose interior is made acid with CO2. Removal of cells with EDTA, pronase E and dispase has similar effects. It does not occur, or is greatly reduced, in cells already rounded up. Substances of molecular weight up to 5000 (e.g. inulin) also cross the cell membrane during this phase. We think that the effect is due to a transient increase in leakiness of the cell during rounding up, possibly due to the detachment of the 'feet' holding the cells onto the substrate. The transient increase in permeability of these cells may be a valuable method of introducing large molecules into them.

Radiolabeling of equine platelets in plasma with 111In-(2-mercaptopyridine-N-oxide) and their in vivo survival.

A method is presented for the in vitro isolation and radiolabeling of equine platelets with the isotope indium 111 (111In: half-life = 2.8 days, gamma = 173 keV, 89%; 247 keV, 94%). The technique described involves complexing 111In with the lipid-soluble chelating agent, 2-mercaptopyridine-N-oxide (merc), in an aqueous medium. 111In-merc platelet-labeling efficiencies in autologous plasma pretreated with or without ferric citrate reagent were 82 +/- 7% and 24 +/- 12%, respectively. Mean intravascular survivals of 111In-merc-radiolabeled platelets in 8 healthy horses according to simple linear, exponential, mean, weighted-mean residual sum of squares analysis, and multiple-hit model were 5.5 +/- 0.49, 3.5 +/- 0.53, 4.5 +/- 0.18, 4.3 +/- 0.65, and 3.6 +/- 0.97 days, respectively.

The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it.

HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not. The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured. In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured. The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr-1, similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain. In the sucrose gradient experiments it was found that [3H]ouabain, digoxin and digitoxin all initially co-distribute with the plasma membrane marker, 5'-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37 degrees, to co-distribute with the lysosomal marker, beta-hexosaminidase. At 2 degrees the ouabain remains co-distributed with the plasma membrane marker. The rate of transfer is estimated to be some 90% hr-1, much faster than previously thought. Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate. These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell. The main barrier for ouabain occurs at a stage later than this. The consequences of this model on other aspects of pump activity is discussed.

Effect of serum on sodium pump activity at various concentrations of internal and external sodium in mouse (3T3) and human (HeLa) cells.

Serum causes a large rise in the ouabain-sensitive K influx in 3T3 and other cells. It has been argued that this is secondary to the increased Na and K leaks which are also caused by serum. We have measured the K influx into 3T3 and HeLa cells at various values of intracellular Na concentration ([Na]i), and from Na-free medium in 1 and 20% (v/v) serum. Raising [Na]i increases the K influx, but saturation does not occur over the range of [Na]i studied. The estimated Km for [Na]i is around 130 mM and the Vmax some 10 times that of the resting K influx at normal (20 mM) [Na]i. Serum stimulates the K influx by a constant proportion at all values of [Na]i studied, and in Na-free medium. These results are inconsistent with previous explanations of the serum stimulation of pumping.

The influence of digoxin antibodies on digoxin disposition and effect: studies in guinea-pigs and HeLa cells.

Pretreatment of guinea-pigs with digoxin-specific Fab (fragment antigen binding) fragments reduced the cardiotoxicity of intravenously infused digoxin (the lethal doses in Fab-treated and control animals were 1.0 and 0.6 mgkg-1, respectively). At death the serum digoxin concentration was elevated 2 fold in the Fab-treated animals, while the tissue concentrations were generally lower. The 30-40% lower cardiac digoxin concentration (seen in whole homogenate and throughout the subcellular fractions examined) was surprising; presumably this reflects a difference from the controls in the proportion of pharmacologically active/inactive digoxin in this organ. Adding digoxin-specific immunoglobulin G or the Fab fragments to HeLa cells before incubation with digoxin, reduced specific digoxin binding (Na pump-bound) slightly more than the non-specific binding. Adding specific antibody after digoxin, however, did not reduce digoxin binding or effect a recovery in Na pump activity. It seems that the protective effect of digoxin-specific antibodies seen in the guinea-pig can to some extent be simulated using HeLa cells. However, this is apparently not so regarding the widely-reported ability of these antibodies to reverse the action of digoxin.

Effect of the serum concentration of the growth medium on the sodium pump site density of cultured HeLa cells.

The density of sodium pump sites in the plasma membrane of cultured HeLa cells has been measured as a function of the serum concentration of the cell growth medium. Growth in media containing increased concentrations of serum (from 1 to 20% v/v) leads to an increase in sodium pump site numbers (as measured by the specific binding of [3H]ouabain) and pump activity (as measured by the ouabain-sensitive 86Rb influx). Time-course studies show that (1) new sodium pump sites first appear some 3-6 h after transfer to medium containing an elevated serum concentration and (2) the serum-mediated increase in new sodium pump sites is completely abolished by the protein synthesis inhibitors, cycloheximide and actinomycin D. These results suggest that de novo protein synthesis is required for the development of the serum response. Preliminary characterization of the serum factor responsible for initiating the synthesis of new sodium pump sites indicates that the activity is associated with a high molecular weight serum fraction (greater than 50000). The different types of interaction seen between the serum effect and other experimental manoeuvres which initiate the synthesis of new pump sites (growth in low-K+ medium, growth in Li+ medium and pre-treatment with exogenous ATP) suggest that there may be more than one pathway for the control of sodium pump site synthesis in cultured HeLa cells.

The effects of chloroquine and other weak bases on the accumulation and efflux of digoxin and ouabain in HeLa cells.

We have studied the effects of the weak bases chloroquine, NH4Cl and amantadine on the handling of certain cardiac glycosides by HeLa cells. When these weak bases are applied acutely to HeLa cells they have only minor effects on the binding of cardiac glycosides to the sodium pumps and on the recovery of pump function following block. When cells are grown in these weak bases there is a variable (10-30%) reduction in pump numbers. This effect is additive to that of chronic treatment with cardiac glycosides. If all sodium pumps are blocked with ouabain, digoxin or digitoxin then recovery of function recovers with a T1/2 of about 7 h (10% h-1); digoxin and digitoxin molecules are excreted at a similar rate but ouabain excretion occurs at a much slower rate (3% h-1). These weak bases greatly slow (x 3) the rate of excretion of digoxin and digitoxin but do not alter that of ouabain. The process affected by chloroquine was estimated to have a T1/2 of 8 h. Cells grown in the presence of cardiac glycosides accumulate large numbers of glycoside molecules; chloroquine, NH4Cl and amantadine increase the accumulation of digoxin and digitoxin and may decrease that of ouabain. Quantitatively these results fit a model whereby cardiac glycosides are accumulated by HeLa cells bound to the sodium pumps, are processed by the lysosomes and then excreted. The results are consistent with a process of internalisation and renewal of sodium pumps by HeLa cells.

Sudden large and periodic changes in heart rate in healthy young men after short periods of exercise.

The instantaneous heart rate and respiratory pattern were recorded immediately after brief periods of exercise in 41 healthy male students. Recordings were taken with the subjects both supine and standing. More than half of these subjects showed oscillatory heart changes when recovering supine but not when standing. During these oscillations the heart rate slowed suddenly by more than 30 beats/min; the oscillations had a period of 4 to 8 seconds, and they continued for half to two minutes. The P waves of the electrocardiogram were decreased during the slowing, consistent with increased vagal activity. When these oscillations occurred they each followed the start of an inspiration with the same latency as in respiratory sinus arrhythmia; unlike respiratory sinus arrhythmia, however, they did not occur after every inspiration but varied from 1:1 to 1:3 oscillations:breaths. They were not usually stopped by breath holding but were reduced or abolished by procedures which reduced venous return. This pattern of oscillations--"vagushalt"--seems to be different from respiratory sinus arrhythmias, and central venous pressure may contribute to the phenomenon. Although it is not widely recognised, vagushalt is probably very common and possibly its occurrence may change in disease.