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Departments of 1 Biomedical Sciences and 2 Agricultural and Biological Engineering, Cornell University, Ithaca, New York 14853; 3 Department of Physiology, University of Munich, D-80336 Munich, Germany; and 4 VERU, Southern Plains Agricultural Research Center, United States Department of Agriculture, College Station, Texas 77845
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Principal cells of the Malpighian tubule of
the yellow fever mosquito were studied with the methods of
two-electrode voltage clamp (TEVC). Intracellular voltage
(Vpc) was 
86.7 mV, and input resistance
(Rpc) was 388.5 k
(n = 49 cells). In six cells, Ba2+ (15 mM) had negligible effects
on Vpc, but it increased
Rpc from 325.3 to 684.5 k (P < 0.001). In the presence of Ba2+, leucokinin-VIII (1 µM) increased Vpc to 101.8 mV
(P < 0.001) and reduced Rpc to
340.2 k (P < 0.002). Circuit analysis yields the
following: basolateral membrane resistance, 652.0 k; apical membrane
resistance, 340.2 k; shunt resistance (Rsh),
344.3 k; transcellular resistance, 992.2 k. The fractional
resistance of the apical membrane (0.35) and the ratio of transcellular
resistance and Rsh (3.53) agree closely with
values obtained by cable analysis in isolated perfused tubules and
confirm the usefulness of TEVC methods in single principal cells of the
intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized
Vpc from 94.3 to 10.7 mV (P < 0.001) and reversibly increased Rpc from 412 to 2,879 k (P < 0.001), effects that were
duplicated by cyanide (0.3 mM). Significant effects of metabolic
inhibition on voltage and resistance suggest a role of ATP in
electrogenesis and the maintenance of conductive transport pathways.
yellow fever mosquito; shunt resistance; potassium channel; barium; leucokinin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MALPIGHIAN TUBULES
of the yellow fever mosquito consist of two types of cells, principal
cells and stellate cells. Principal cells are the most numerous. They
are large, fusiform cells that may envelop the tubule lumen. Also known
as primary cells, they power the secretion of Na+ and
K+ from the hemolymph into the tubule lumen via active
transport mechanisms (4). Stellate cells comprise about
18% of the total cell population (3, 22). They are
thought to provide the pathway for transepithelial movement of
Cl
in Malpighian tubules of the fruit fly
(17). However, our laboratory has evidence for the passage
of Cl through the paracellular pathway in Malpighian
tubules of the yellow fever mosquito (18). The
paracellular pathway in insect Malpighian tubules is defined by septate
or continuous junctions rather than tight junctions (9, 16, 24,
29).
We describe here the first exploration of single principal cells in the
intact tubule with the methods of two-electrode voltage clamp (TEVC).
Using Ba2+ to block basolateral K+ conductance
and leucokinin-VIII to increase paracellular shunt Cl
conductance, we obtained estimates of the resistances of the basolateral and apical membranes of principal cells and the resistance of the shunt pathway. Good agreement of these estimates with those obtained by cable analysis (CA) in isolated perfused tubules
(19) validates the use of TEVC in single principal cells.
Moreover, metabolic inhibition with dinitrophenol (DNP) or cyanide
(KCN) reduces the intracellular voltage of principal cells
(Vpc) to values only 11% of control and
increases cell input resistance (Rpc) to
values more than 700% of control. Full reversibility of the effects of
metabolic inhibition on voltage and resistance with the speed of the
bath change (seconds) suggests high turnover rates of ATP and the
central role of this nucleotide in electrogenesis in Malpighian tubules.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mosquitoes and Malpighian tubules. The mosquito colony was maintained as described by Pannabecker et al. (18). On the day of the experiment a female mosquito (3-7 days post-eclosion) was cold anesthetized and decapitated. A Malpighian tubule was removed under Ringer solution from its attachment to the gut and transferred to a Lucite perfusion chamber with a filling volume of 0.5 ml. The bottom of the chamber was covered with a thin layer of black dissecting wax, to which Malpighian tubules readily adhere. The tubules were viewed from above with a stereoscope microscope at ×50 magnification (Wild, Heerbrugg, Switzerland). A principal cell was selected near the blind end of the tubule and impaled with two conventional microelectrodes for TEVC studies.
Ringer solution and drugs. Ringer solution contained the following (in mM): 150 NaCl, 25 HEPES, 3.4 KCl, 1.8 NaHCO3, 1 MgCl2, 1.7 CaCl2, and 5 glucose. The pH was adjusted to 7.1 with NaOH. The osmolality of Ringer was 320 mosmol/kgH2O. High-K+ solution contained 34 mM K+, substituting K+ for Na+ in equimolar quantities.
To block the K+ conductance of the basolateral membrane of principal cells, we used BaCl2 at concentrations up to 15 mM. In these experiments, the control Ringer solution contained a concentration of mannitol equivalent to the osmolytes of BaCl2. Synthetic leucokinin-VIII was a gift from Mark Holman and Ron Nachman (Texas A&M University). All agents were added to the peritubular Ringer solution. Normally, the bath volume was ~250 µl, as Ringer solution flowed through the perfusion chamber at a rate of 6.5 ml/min. On the assumption that the bath change can be described by first order kinetics, it takes only 10 s to replace 99% of the bath volume. Rapid bath changes were desirable in evaluations of the effects of barium, DNP, and KCN on voltage and resistance. However, bath flow was stopped before adding leucokinin-VIII to conserve peptide.Electrophysiological studies.
All electrophysiological measurements were made in nonperfused tubules
resting on the bottom of the perfusion bath (Fig.
1A). Tubules were ~3.5 mm
long. A principal cell, ~0.5 mm from the blind end of the tubule, was
selected for impalement with current and voltage electrodes. The
placement of microelectrodes ~10 length constants from the open end
of the tubule, reduced short-circuiting into the bath of the current
injected into the cell (1, 19). Impalement of the
principal cell near its center yielded the most stable current and
voltage recordings.
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when filled with 3 M KCl. One
electrode served to record the Vpc, and the
other served to inject current when measuring
Rpc. The electrodes were bridged to the
measuring and clamp circuits using Ag/AgCl junctions that were prepared
by first degreasing the Ag wire with alcohol, and then by Cl-plating it
in 0.1 M HCl for 20 min at a current of 50 µA. The bath was grounded
with a 4% agar bridge containing Ringer solution.
For voltage and current recording from principal cells, we used the
GeneClamp model 500 voltage and patch clamp amplifier (Axon
Instruments, Foster City, CA) equipped with head stages for TEVC
experiments in oocytes: head stage HS-2A gain 10MGU for current
injection and head stage HS-2A gain 1LU for voltage recording. The
voltage clamp was only engaged for measurements of
Rpc, which was evaluated from the current
changes accompanying four or more 11-ms hyperpolarizing voltage clamp
steps of 10-30 mV each. Clampfit (pClamp 6, Axon Instruments,
Foster City, CA) was used to produce current-voltage (I-V)
plots from which resistance was determined from linear fits to the data.
All current and voltage data were displayed on an oscilloscope (Iwatsu,
Japan) and on a strip chart recorder (model BD 64; Kipp and Zonen,
Crown Graphic). Data were also collected in digital form with the aid
of a Macintosh computer (7300/200) equipped with data acquisition
hardware and software (Multifunction I/O Board PCI-1200 and Signal
Conditioning and Termination Board Model SC-2071; LabView for
Macintosh, version 4.1; National Instruments Manufacturer, Austin, TX).
Circuit analysis.
Since transepithelial electrolyte secretion in Malpighian tubules of
Aedes aegypti is electrogenic (4, 5, 28),
transepithelial transport of ions can be modeled with the electrical
equivalent circuit shown in Fig. 1B. In brief, the active
transport pathway, taken by Na+ and K+ through
principal cells, consists of electromotive forces (E) and
the resistance (R) of the basolateral (bl) and apical (a) membranes. A shunt pathway for Cl consists of the single
resistance, Rsh, located outside principal cells. Transcellular and paracellular pathways are electrically coupled
such that cationic current through principal cells equals anionic
current through the shunt (Fig. 1B).
, is constant and subtracted in all resistance measurements. The
resistance of fluid secreted into the tubule lumen
(Rlumen) is in contact with the peritubular
Ringer bath via the open end of the tubule (Fig. 1A). Since
Rlumen is about 2,000 times greater than the Rsh (1, 19), the fraction
of current that crosses the apical membrane into the tubule lumen does
not short-circuit through the open end of the tubule, but returns to
ground via the nearest shunt (Rsh). Hence,
Rlumen can be neglected in the circuit analysis, which reduces the electrical model to two parallel pathways for injected current: one pathway across the basolateral membrane of the
principal cell, the other across the apical membrane and the shunt
(Fig. 1D).
We used two assumptions to obtain numerical estimates for the
resistances of cell membranes and the shunt. The first assumption, the
"barium assumption," states that Ba2+ increases the
resistance of the basolateral membrane to such high values that
measurements of Rpc(Ba) approach the sum of the apical membrane resistance and the Rsh. The
second assumption, the "leucokinin assumption," states that in the
presence of Ba2+ the application of leucokinin-VIII lowers
the Rsh resistance to such low values that
measurements of Rpc(Ba+LK) approach the
resistance of the apical membrane (Eq. 1). Thus
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(1) |
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(2) |
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(3) |
Statistical evaluation of data. Each cell was used as its own control so that the data could be analyzed for the difference between paired samples, control vs. experimental (paired Student's t-test).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Control values of basolateral membrane voltage and input resistance
of principal cells.
Under control conditions, the average Vpc was
86.7 ± 1.2 mV in 49 principal cells, each from a different
Malpighian tubule. The Rpc was determined by
voltage clamping of Vpc to a series of
potentials by current injection into the cell. Figure
2 shows representative current traces and
the corresponding I-V relationships for control conditions
and after addition of 5 mM Ba2+ to the peritubular Ringer
bath. Charging of membrane capacitance was essentially complete within
the pulse duration of 11 ms (Fig. 2). The current overshoots, lasting
for about 2 ms at the onset and termination of the pulse, reflect
mostly the time constants of the clamp circuit. A plot of clamp voltage
vs. current yields I-V relationships that reveal small
deviations from linearity with increasing conductance at
hyperpolarizing voltages. For the principal cell shown in Fig. 2,
Rpc estimated in the vicinity of zero current
(open-circuit voltage) was 371.4 k under control conditions.
Ba2+ did not alter the deviation from linearity of the
I-V relationship. It did, however, decrease the slope of the
I-V plot to reflect the increase in
Rpc to 740.4 k. In subsequent experiments,
Rpc was measured from a linear fit to four
hyperpolarizing clamp steps of 10-20 mV each.
Rpc was on average 388.5 ± 13.4 k for
49 principal cells (tubules).
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Effects of K+ and Ba2+ on voltage and
resistance.
Previous studies suggested that K+ uptake across the
basolateral membrane of principal cells is passive, presumably through K+ channels (4, 5). Thus it was of interest to
analyze the response of the basolateral membrane to elevations of bath
K+ concentration and to blockage of K+ channels
with Ba2+. Figure 3 shows a
typical experiment. After electrode impalements, Vpc stabilized at a value of 98 mV;
Rpc at that time was 270 k. Increasing bath
K+ concentration from 3.4 to 34 mM decreased
Vpc and
Rpc to 52 mV and 180 k,
respectively. Both Vpc and
Rpc returned to control values
upon returning bath [K+] to the normal concentration of
3.4 mM. Depolarization and repolarization of Vpc
were as fast as the rate of bath change. On average, a 10-fold increase
in peritubular [K+] caused the significant
(P < 0.001) depolarization of
Vpc from 86.7 ± 4.5 to 47.0 ± 3.2 mV and a significant (P < 0.001) decrease in
Rpc from 416.2 ± 40.1 to 257.2 ± 21.8 k (mean ± SE, n = 6 principal cells; 6 tubules).
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Estimates of membrane and shunt resistances.
As shown in Fig. 1D, the input resistance
Rpc is comprised of three components,
Rbl, Ra, and
Rsh, which can be estimated using Ba2+ and leucokinin-VIII, as described in the
MATERIALS AND METHODS. Data from six cells are summarized
in Fig. 5. Under control conditions, Vpc was 73.2 ± 5.6 mV, and the
Rpc was 325.3 ± 31.0 k (Table 1). The addition of 15 mM
Ba2+ to the peritubular bath solution had little effect on
Vpc, but it significantly increased
Rpc to 684.5 ± 46.5 k
(P < 0.001). The subsequent application of
leucokinin-VIII (106 M) in the presence of
Ba2+ significantly (P < 0.002)
hyperpolarized the cell to 101.8 ± 6.9 mV, whereas
Rpc significantly (P < 0.003)
decreased to 340.2 ± 46.6 k. These changes in
Vpc and Rpc induced by
Ba2+ and leucokinin-VIII were reversible in the stepwise
return to control Ringer solution, consistent with specific as well as
independent effects of Ba2+ and leucokinin-VIII on the
basolateral K+ channels and the shunt, respectively (Fig.
5; 14).
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for
the apical membrane, 652.0 k for the basolateral membrane, and 344.3 k for the shunt (Eqs. 1-3). In Table 1 these
resistances are compared with those determined by cable analysis (CA)
in isolated perfused Malpighian tubules. The comparison must be
relative because resistances determined by CA are normalized to tubule
length, which cannot be done in TEVC studies of a principal cell.
However, taking ratios cancels normalization, thereby allowing a
quantitative comparison of the two methods (Table 1). Accordingly, the
ratio of transcellular and shunt resistance is 3.33 by CA and 3.53 by TEVC, and the fractional resistance of the apical membrane of the
principal cell is 0.32 by CA and 0.35 by TEVC.
Effects of metabolic inhibitors.
In each of three cells studied, DNP reversibly affected intracellular
voltage and input resistance (Table 2).
In the presence of DNP, Vpc went from 94.3 to
10.7 mV, whereas Rpc increased sevenfold from
411.7 to 2,879.0 k in three cells. Similar results were obtained
using KCN (0.3 mM), another inhibitor of ATP synthesis (Table 2). In
the presence of cyanide, Vpc went from 85.6 to 10.0 mV, whereas Rpc increased eightfold from
389.0 to 3,226.9 k in seven cells. The voltage and resistance
changes induced by DNP and KCN were highly significant (Table 2). What
is astonishing is the rapidity of the on/off effect of
metabolic inhibition. As soon as DNP or KCN flowed into the peritubular
bath, voltage decreased and resistance increased with the speed of the
bath change. Likewise, washout of DNP or KCN restored control values of
voltage and resistance with the speed of the solution change.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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TEVC experiments in principal cells of the Malpighian tubule. TEVC is a valuable method for the study of ion movement across the Xenopus oocyte membrane. For other systems, in particular epithelial cells in situ, this technique has hitherto not been applied. The major obstacle is the commonly small size of cells which precludes impalement with two electrodes. Size is not a limitation in principal cells of the Malpighian tubule of A. aegypti. We found that impalements by two electrodes were durable for several hours, in one case over 4 h; they survived repeated solution changes and repeated voltage perturbations for the examination of I-V relationships (Figs. 2 and 3).
Current-voltage relationships in principal cells.
The average voltage measured in principal cells,
Vpc = 86.7 mV, is the voltage across the
basolateral membrane (Vbl). It is also the
voltage across the apical membrane in series with the shunt pathway
(Va + Vt; Fig. 1).
In the present study, Vbl was significantly
greater than 66.2 mV, measured previously in nonperfused Malpighian
tubules (23), and significantly greater than 58.0 mV,
measured in isolated perfused tubules (18). The basolateral membrane voltage of Aedes Malpighian tubules is
known to vary widely with rates of transepithelial transport. For
example, Vbl is 77 mV under control conditions
when tubules secrete fluid at a rate 0.8 nl/min; but in the presence of
cAMP, Vbl is only 24 mV when fluid secretion
rises 250% to 2.8 nl (5, 27). Thus variations of
Vbl reflect the functional range of the
basolateral membrane between antidiuretic and diuretic transport rates.
Furthermore, the resistance of the basolateral membrane is twice as
great as that of the apical membrane, yielding voltage drops (current
times resistance) across the basolateral membrane twice as great as those across the apical membrane (Table 1; Ref. 4, 23).
140 mV. A
systematic analysis, attempting to associate this increase with a
particular ion pathway, was not the object of the present study
exploring the feasibility of TEVC methods in principal cells of the
Malpighian tubule. For this reason we confined measures of resistances
to the immediate neighborhood of open-circuit voltages, which in 49 principal cells yielded an average input resistance of 388.5 k under
control conditions.
Estimates of membrane and shunt resistances.
In the present study, membrane and shunt resistances were estimated
with the aid of two assumptions, the barium assumption, and the
leucokinin assumption (see MATERIALS AND METHODS). In the
strictest sense, the barium assumption is not fulfilled since Ba2+ may not block all conductances of the basolateral
membrane, but it must block the major, dominant conductance of the
basolateral membrane. Since Rpc is the parallel
resistance of the basolateral membrane and the sum of the resistances
of the shunt and apical membrane (Fig. 1D), the basolateral
membrane resistance must increase 43-fold in order for
Rpc to increase from 321.3 to 682.3 k in the
presence of 15 mM Ba2+ (Fig. 4). Thus a 43-fold increase in
basolateral membrane resistance meets the barium assumption (Fig.
1D).
· cm in isolated perfused Malpighian
tubules (18, 19). Such a large decrease in the Rsh meets the leucokinin assumption for
estimates of the apical membrane resistance. Accordingly,
Rpc in the presence of both Ba2+ and
leucokinin-VIII approaches the resistance of the apical membrane: 340.2 k. It follows that the Rsh is 344.3 k, and
the Rbl is 652.0 k (Table 1, Eqs.
1-3, Fig. 1D). The ratio of the transcellular and
shunt resistance is 3.53, and the fractional resistance of the apical
membrane is 0.35 (Table 1). Remarkably similar ratios, 3.33 and 0.32, respectively, were obtained using 1) a different experimental method, CA in isolated perfused Malpighian tubules (19); 2) a variation of the Yonath-Civan method
to estimate the shunt resistance (30); and 3) a
different agent, DNP, to distinguish between transcellular and
paracellular pathways (Table 1). The excellent agreement between
resistance ratios measured by TEVC methods (and the use of
Ba2+ and leucokinin-VIII) and CA of in vitro perfused
Malpighian tubules (and the use of DNP) confirms the validity of
voltage clamp studies in single principal cells of Malpighian tubules
and bolsters confidence in both methods.
Electrical coupling of principal cells. Validation of the methods of TEVC in principal cells of Malpighian tubules allowed a preliminary estimate of the degree of electrical coupling (Table 1). The transepithelial conductance in the isolated perfused Malpighian tubule is the sum of the shunt conductance (1/Rsh) and the transcellular conductance (1/Rcell), where Rcell is the sum of Ra and Rbl. Using the data of Table 1, the transepithelial conductance in isolated perfused tubules is 87.7 µS/cm tubule length. TEVC studies (Table 1) yield a transepithelial conductance of 3.91 µS/principal cell, or 22.4 cells for a tubule 1 cm long (Table 1). An actual count yields ~125 principal cells/cm tubule length (21). It follows that the input conductance of a principal cell does not reflect a single cell but several principal cells, 5.6 cells on average, that must be electrically coupled.
The basolateral membrane of principal cells. The present study confirms previous data (23), indicating a dominant K+-conductive pathway in the basolateral membrane of principal cells. With the possible exception of Drosophila (12), most Malpighian tubules offer such a K+ conductance in membranes facing the hemolymph (10, 11, 13, 15, 26). Moreover, intracellular K+ is close to electrochemical equilibrium with extracellular K+ in Malpighian tubules of A. aegypti and other insects (10, 13, 20, 26). In the present study, the maximal inhibition of the basolateral membrane K+ conductance in control Ringer solution occurs at a Ba2+ concentration of 1.5 mM (Fig. 4). The basolateral membrane voltage may hyperpolarize in the presence of Ba2+, but it never depolarizes (Figs. 2-4), which is opposite to the Ba2+ response of most other cells of vertebrate origin, where the block of K+ conductance depolarizes membrane voltages toward zero (2). The effect of Ba2+ hyperpolarizing membrane voltages is not uncommon in cells of insect origin. Ba2+ hyperpolarizes the basolateral membrane voltage of locust hindgut epithelial cells (7) and midgut epithelial cells of the larval tobacco hornworm (13).
In the case of principal cells of Aedes Malpighian tubules, the essential polarizing component of the basolateral membrane is not a K+ diffusion potential, but rather the electrogenic proton pump located in the apical membrane (6, 8). As illustrated in the transport model of Fig. 6, this proton pump is a V-type ATPase that extrudes H+ into the tubule lumen (or the microenvironment of apical microvilli) at the expense of metabolic energy. Active H+ transport into the tubule lumen polarizes the apical membrane (cell negative) with respect to the luminal fluid to values in excess of 110 mV (Fig. 6, Ref. 4, 5). Current returning to the cytoplasmic side of the pump, via the shunt and the basolateral membrane, couples the electrical potential of the apical membrane to the basolateral membrane, where it provides the electromotive force for the entry of K+ into the cell. K+ entry into the cell depolarizes the basolateral membrane. Thus, blocking K+ influx with Ba2+ hyperpolarizes the membrane (Fig. 3). The opposite is observed after increasing K+ flux into the cell by elevating bath K+ concentration, together with a decrease in input resistance (Fig. 3). The reduced efficacy of Ba2+ to block basolateral membrane conductance in the presence of high K+ concentrations suggests competition between K+ and Ba2+ for K+ channels, as in neural and epithelial membranes of vertebrate origin (2, 25).
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, from the peritubular medium to the lumen
through the shunt (Fig. 6).
Metabolic inhibitors. In a previous study of isolated perfused Malpighian tubules (19), we learned that DNP reversibly abolished basolateral and apical membrane voltages of principal cells to values close to 0 mV while increasing the transepithelial resistance (Rt) by 47% (Fig. 1B). We interpreted these changes to indicate the inhibition of active transport through principal cells. Moreover, we assumed that this inhibition increases the transcellular resistance (Rbl + Ra) such that measurements of the Rt approach the Rsh (Table 1, Fig. 1). The present study using TEVC allowed a test of this assumption.
Both DNP and KCN depolarized the intracellular voltage of principal cells to10 mV while increasing the Rpc seven-
to eightfold (Table 2). Such a large increase in
Rpc confirms our previous assumption regarding
the increase in transcellular resistance after metabolic inhibition by
DNP (19). Moreover, in the present study,
Rpc in the presence of DNP is largely the
parallel resistance of basolateral and apical cell membranes (Fig.
1D). Thus the resistances of both basolateral and apical
membranes during metabolic inhibition can be calculated from
Rpc in the presence of DNP or KCN, since it is
known that DNP increases the fractional resistance of the apical
membrane from 0.32 to 0.57 (19). These calculations show that DNP causes the basolateral membrane resistance to increase from
652 to 5,038 k, and the apical membrane to increase from 340 to
6,718 k. Similar increases take place in the presence of KCN. These
large increases in basolateral and apical membrane resistance suggest
that conductive pathways in both membranes are shutting down during
metabolic inhibition, reducing or preventing movement of ions into and
out of the cell. Intracellular ion homeostasis would thus be preserved,
allowing transport to spring back again after metabolism is restored.
Full recovery of voltage and conductance after washout of metabolic
inhibitors is consistent with this interpretation of the data (Table
2). DNP is known to collapse proton gradients across mitochondrial
membranes, and cyamide is an inhibitor of cytochrome c
oxidase. Since both DNP and KCN inhibit ATP synthesis at different
sites of oxidative metabolism, their effects on voltage and resistance
most likely reflect the inhibition of ATP synthesis and intracellular
ATP depletion. Accordingly, the effects of metabolic inhibition on
voltage and resistance suggest high turnover rates of ATP and a role of
ATP in maintaining conductive transport pathways in principal cells.
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ACKNOWLEDGEMENTS |
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We thank Mark Baustian for editorial improvements.
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FOOTNOTES |
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We thank the National Science Foundation for enabling this study (IBN 9604394).
Address for reprint requests and other correspondence: K. W. Beyenbach, Dept. of Biomedical Sciences, VRT 8014, Cornell Univ., Ithaca, NY 14853 (E-mail: kwb1{at}cornell.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 February 2000; accepted in final form 31 May 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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