PubMed Health⌕ Search

Biomedical subjects

D F Moffett

Publications and source records attributed to D F Moffett.

At least 19 recordsLinked to original sources
PubMed2005

Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin.

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.

Action Potentials↗
PubMed2004

The transepithelial voltage of the isolated anterior stomach of mosquito larvae (Aedes aegypti): pharmacological characterization of the serotonin-stimulated cells.

The lumen-negative transepithelial voltage (V(te)) of the isolated and perfused anterior stomach of mosquito larvae (Aedes aegypti) was studied with a 'semi-open' preparation in which one end of the gut was ligated onto a perfusion pipette and the other end remained open to the bath. All experiments were performed with serotonin-stimulated preparations. V(te) was abolished after addition of 2.5 mmol l(-1) dinitrophenol and depended on the presence of Cl(-). Na(+) substitution experiments showed that a major part of V(te) depended on the presence of this cation in the hemolymph side of the epithelium. Addition of 10 micro mol l(-1) concanamycin (78+/-6% inhibition) or 2.5 mmol l(-1) ouabain (15+/-2% inhibition) to the bath partially inhibited V(te). DPC (0.5 mmol l(-1)) or DIDS (0.1 mmol l(-1)) reduced V(te) when applied to the hemolymph side of the epithelium (to 49+/-8% or 78+/-3% of the control, respectively). When present on both sides of the epithelium, these inhibitors caused further V(te) reductions (to 23+/-4% or 35+/-4% of the control, respectively). Hemolymph-side furosemide (0.1 mmol l(-1)) or BaCl(2) (5 mmol l(-1)) reduced V(te) by 13+/-3% or 23+/-4% of the control, respectively. When applied to the hemolymph side of the epithelium, amiloride (0.2 mmol l(-1)) significantly decreased V(te) by 35+/-6% of the control, whereas the drug caused no further effect when it was subsequently also applied to the luminal side of the epithelium. The above results are the basis for an extended model for the cellular mechanisms of NaHCO(3) secretion/HCl absorption involved in alkalization of the anterior stomach of mosquito larvae.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility.

The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3- secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1-5) all affected Vte in almost identical ways, causing a 10-15% reduction of the voltage at 10(-7) mol l(-1). A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10(-6) mol l(-1), neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vte deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10(-8) to 10(-6) mol l(-1). These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

Aedes↗
PubMed2000

The electrical properties of the anterior stomach of the larval mosquito (Aedes aegypti).

The electrical properties of the anterior stomach of the larval mosquito (Aedes aegypti) were determined. At late times after cannulation, the intraluminal space constant was 936 microm, which is almost as long as the isolated tissue itself. At this time, the resistance of the apical cell membranes dominates the transcellular resistance; it is approximately 14 times the resistance of the basal cell membrane. Two physiologically distinct epithelial cell types were identified. One type has a stable basal potential of approximately 65 mV and responds to 5-hydroxytryptamine with hyperpolarization. The second cell type initially shows a basal potential of 100 mV. However, this basal potential decays in the first few minutes in parallel with the decay of the transintestinal potential. This latter cell type does not respond to 5-hydroxytryptamine.

Aedes↗
PubMed1999

The anterior and posterior 'stomach' regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine.

The 'stomach' region of the larval mosquito midgut is divided into histologically distinct anterior and posterior regions. Anterior stomach perfused symmetrically with saline in vitro had an initial transepithelial potential (TEP) of -66 mV (lumen negative) that decayed within 10-15 min to a steady-state TEP near -10 mV that was maintained for at least 1 h. Lumen-positive TEPs were never observed in the anterior stomach. The initial TEP of the perfused posterior stomach was opposite in polarity, but similar in magnitude, to that of the anterior stomach, measuring +75 mV (lumen positive). This initial TEP of the posterior stomach decayed rapidly at first, then more slowly, eventually reversing the electrical polarity of the epithelium as lumen-negative TEPs were recorded in all preparations within 70 min. Nanomolar concentrations of the biogenic amine 5-hydroxytryptamine (5-HT, serotonin) stimulated both regions, causing a negative deflection of the TEP of the anterior stomach and a positive deflection of the TEP of the posterior stomach. Phorbol 12,13-diacetate also caused a negative deflection of the TEP of the anterior stomach, but had no effect on the TEP of the posterior stomach. These data demonstrate that 5-HT stimulates region-specific ion-transport mechanisms in the stomach of Aedes aegypti and suggest that 5-HT coordinates the actions of the Malpighian tubules and midgut in the maintenance of an appropriate hemolymph composition in vivo.

Aedes↗
PubMed1998

Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance.

Real-time biospecific interactions of immunogens, measured via BIAcore, were used to verify qualitatively a biosensor design which relies on analyte binding competition reactions to open cross-linked receptor channels. The complexes of importance are: (1) cardiac troponin I (TnI) and monoclonal mouse anti-TnI IgG mAb 265, (2) TnI and bispecific antibodies (BsAbs) which on one end recognize TnI while the other end recognizes nicotinic acetylcholine receptors (nAChRs), (3) nAChRs and rat anti-nAChR IgG mAb 148, (4) nAChRs and BsAbs, (5) nAChRs and Fab'148-TnI biopolymers, and (6) mAb 265 and Fab-TnI biopolymers. A commonly used sensor chip, CM5, was employed to immobilize TnI by covalent amine coupling, while bilayer membrane-associated protein, nAChR, was noncovalently sequestered on a HPA sensor chip via hydrophobic adsorption of membrane lipids. The epitopes of membrane-bound nAChRs were still available to immunogens after being immobilized. Kinetic rate constants and affinities of these systems were calculated from BIAcore sensorgrams. The order of magnitude for dissociation rate constants of the BsAb/TnI linker complex and biopolymer/mAb 265 complex is 10(-2) s-1, which provides an opportunity for competitive binding of free analyte in the sensing systems.

Animals↗
PubMed1997

Kinetic modeling and analysis of a vesicle system for immunosensor development.

A novel mechanism is presented for immunosensor development that uses an immunological competition reaction in a vesicle system. This system consists of a suspension of reconstituted vesicles, channel agonist, protein linker to block the channels, voltage sensitive dye and analyte to be detected. In the proposed mechanism analyte serves a catalytic role as individual analytes competitively displace multiple channel linkers through association with one channel, dissociation and new associations with other channels. When one channel opens on a vesicle a permanent Nernst potential develops for that vesicle leading to fluorescence of voltage sensitive dyes. The time constant of the redistribution from linker-channel form to analyte-channel form is 0.92/k4 (k4 is the off-rate constant for the analyte-channel association) in the region of analyte concentrations less than 10(-9) M. Kinetic analyses show that several factors, including concentration of analyte or linker, number of channels per vesicle, on-rate or off-rate constant of the linker-channel and on-rate constant of analyte-channel complexes have significant effects on the minimum signal response time.

Antigen-Antibody Reactions↗
PubMed1995

A highly stable and selective biosensor using modified nicotinic acetylcholine receptor (nAChR).

Methods for developing stable, sensitive and selective bilayer lipid membrane (BLM)-based biosensors are discussed. Stable BLMs were formed over micromachined polyimide apertures. Selective sensors were made by incorporating nicotinic acetylcholine receptors (nAChRs) modified with bispecific antibodies (BsAbs). When two BsAbs, attached to one nAChR, encounter antigen (Ag), channels are blocked. Sensitivity to single Ag molecules would be possible by monitoring closure of individual nAChRs.

Animals↗
PubMed1992

Driving forces and pathways for H+ and K+ transport in insect midgut goblet cells.

In the midgut of larval lepidopteran insects, goblet cells are believed to secrete K+; the proposed mechanism involves an electrogenic K+/nH+ (n > 1) antiporter coupled to primary active transport of H+ by a vacuolar-type ATPase. Goblet cells have a prominent apical cavity isolated from the gut lumen by a valve-like structure. Using H(+)- and K(+)-selective microelectrodes, we showed that electrochemical gradients of H+ and K+ across the apical membrane and valve are consistent with active secretion of both ions into the cavity and that the transapical H+ electrochemical gradient, but not the transapical pH gradient, is competent to drive K+ secretion by a K+/nH+ antiporter. We used 10 mmol l-1 tetramethylammonium ion (TMA+) as a marker for the ability of small cations to pass from the gut lumen through the valve to the goblet cavity, exploiting the high TMA+ sensitivity of 'K(+)-sensitive' microelectrodes. These studies showed that more than half of the cavities were inaccessible to TMA+. For those cavities that were accessible to TMA+, both entry and exit rates were too slow to be consistent with direct entry through the valves. One or more mixing compartments appear to lie between the lumen bathing solution and the goblet cavity. The lateral intercellular spaces and goblet cell cytoplasm are the most likely compartments. The results are not consistent with free diffusion of ions in a macroscopic valve passage; mechanisms that would allow K+ secreted into the goblet cavity to exit to the gut lumen, while preventing H+ from exiting, remain unclear.

Animals↗
PubMed1990

Basal membrane uptake in potassium-secreting cells of midgut of tobacco hornworm (Manduca sexta).

Basal membrane voltage (Vb), intracellular K+ activity [(K+)i], and short-circuit current (Isc) were measured in isolated posterior midguts of Manduca sexta wherein Isc is a measured of active secretion of K+ from blood into lumen. When bathed in 32 mM K+ and exposed to 100% O2, average values were Isc = 244 microAmp/cm2, Vb = -33.1 mV, and (K+)i = 88.6 mM. The electrochemical gradient across the basal membrane (d mu) averaged +5.8 mV (a gradient favorable for K+ entry). Exposure to 5% O2 led to a new steady state in which Isc = 71 microAmp/cm2, Vb = -18.7 mV, and (K+)i = 99.4 mM. During hypoxia, d mu averaged -9.9 mV (a gradient unfavorable for K+ entry). When the external bathing solution was 10 mM K+, comparable values were, for 100% O2, Isc = 139 microAmp/cm2, Vb = -56.1 mV, (K+)i = 72.2 mM, and d mu = +3.6 mV, and in 5% O2 the values were Isc = 28.3 microAmp/cm2, Vb = -43.7 mV, (K+)i = 76.1 mM, and d mu = -10.2 mV. The failure of cellular K+ to fall during prolonged hypoxia is evidence for a thermodynamically active basal K+ uptake process.

Animals↗
PubMed1989

Active chloride transport in isolated posterior midgut of tobacco hornworm (Manduca sexta).

The short-circuited posterior midgut of larval tobacco hornworm (Manduca sexta) actively transports Cl- from lumen to hemolymph as measured by unidirectional fluxes of 36Cl-. Potentials and Cl- activities in cytosol and goblet cavity were measured using double-barreled Cl--selective microelectrodes. In the short-circuited tissue, the goblet cavity was electrically positive to the bathing solution, and Cl- activity was below electrochemical equilibrium with luminal fluid. The cytosol was electrically negative to the bathing solution, and Cl- activity was above electrochemical equilibrium. Thus Cl- is pumped from goblet cavity into cell. Although the Cl- that is pumped into the cell can cross the basal membrane, its Cl- conductance is quite low. The Cl- conductance is also quite low in apical membranes of columnar cells. Depression of intracellular Cl- after exposure of the luminal side to high HCO-3 suggested that these membranes have a Cl- for HCO-3 exchange mechanism. The paracellular pathway for Cl- comprises approximately 10% of the total transepithelial conductance.

Animals↗
PubMed1985

Barium modifies the concentration dependence of active potassium transport by insect midgut.

The rate of active K+ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K+] over the range 20 to 70 mM K+ in the absence of any divalent cation. Addition of Ba++ to the hemolymph (K+ uptake) side introduces a linear component to the concentration dependence, such that active K+ transport is decreased at [K+] of 55 mM or less, but increased transiently at higher [K+]. As [Ba++] is increased over the range 2 to 8 mM the linear component increases and the saturating component decreases; in 8 mM Ba++ the concentration dependence is dominated by the linear component. The effect of Ba++ cannot easily be accounted for by simple competition with K+ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K+ transport possesses a substantial linear component in solutions containing 5 mM Ca++ and 5 mM Mg++ (the alkali earth metal concentrations of standard lepidopteran saline).

Animals↗
PubMed1980

Voltage-current relation and K+ transport in tobacco hornworm (Manduca sexta) midgut.

Voltage-current curves for the isolated midgut of the tobacco hornworm were determined by transient and steady voltage clamping over the range of 200 to -200 mV. Over this range the transient method yields a linear relation while the steady method usually yields a curve consisting of two lines of differing slope which intersect at zero voltage. The difference between the results of the methods is due to a slow decline in total conductance which accompanies steady voltage clamping. Holding the midgut at short circuit increases the total conductance of the tissue in a manner consistent with increasing shunt conductance; this effect was seen in both diet-reared and leaf-reared animals. When potassium transport is inhibited by substitution of choline or sodium for potassium in bathing solution the total conductance decreases and the voltage-current curve intersects the normal curve in the hyperpolarizing region. Applying a simple equivalent circuit analysis to the results from partial or total potassium replacement suggests that the electromotive force of the potassium transport system is of the order of 140-190 mV. The conductance decrease during inhibition of potassium transport by transient anoxia is of similar magnitude, suggesting that a major effect of metabolic inhibition is to decrease the active conductance of the potassium transport pathway.

Animals↗
PubMed1980

Temperature coefficients for the oxidative metabolic responses to electrical stimulation in cerebral cortex.

Temperature coefficients of both cat and toad brain have been calculated for the active metabolic state induced by electrical stimulation. Values are higher than most of the values previously reported for "rest" metabolism, whether calculated from Arrhenius plots or from linear graphs. Relative rates of oxidative metabolism were obtained by measuring the time course of the transient changes in NADH fluorescence and cytochrome aa3 absorption by reflectance techniques directly from the surface of the exposed cat cerebral cortex in vivo and from the isolated intact toad brain mounted in a cuvet. These findings demonstrate that such optical methods accurately record metabolic processes.

Animals↗
PubMed1980

Coupling between oxidative metabolism and active transport in the midgut of tobacco hornworm.

Active K transport (Isc) in the midgut of tobacco hornworm Manduca sexta has been shown to be highly dependent on oxidative metabolism. However, the oxygen consumption rate (rO2) was not altered by conditions that drastically affect Isc. Respiration was normally maximal, inasmuch as uncouplers did not increase rO2. This rate could be maintained without any added substrate probably by oxidation of endogenous substrates. Additional succinate increased rO2 by 17%. Simultaneous monitoring of Isc and the redox level of the respiratory chain components demonstrated that 1) succinate (5 mM) reduced all the respiratory enzymes while increasing Isc by 17%; 2) sesamol (5 mM), a mitochondrial uncoupler, reoxidized all respiratory enzymes and inhibited Isc by about 50%; 3) cyanide (1 mM) fully reduced the cytochromes and completely inhibited Isc. These redox responses indicate that the mitochondria in this tissue are normally coupled, even if respiration is maximal and is not modulated by active transport. Mitochondria isolated from the midgut show coupling and respiratory control by ADP, appearing to behave like mitochondria from other tissues. Therefore, a cytoplasmic constraint must exist in this tissue that continually elicits an unmodulated maximal respiratory rate.

Animals↗
PubMed1976

Response of toad brain respiratory chain enzymes to ouabain, elevated potassium, and electrical stimulus.

Spectrophotometric and fluorometric techniques were used to monitor the proportion of reduced to oxidized cytochrome (cyt) and levels of reduced pyridine nucleotide in preparations of whole toad brain in vitro. In resting, well-oxygenated brain, levels of reduction for cyt a3 ranged between 5% and 23%; for cyt a, 17-23%; for cyt c, 18-32%, and for cyt b, 25-42%. These levels of reduction cannot be due to functional hypoxia since hemoglobin in resting brains is 100% oxygenated. In brains treated with 10(-4) M ouabain, stimulant of brain respiration, the cytochromes first become more oxidized, then more reduced; ultimately there is a tendency to return to the initial levels of reduction. In brains bathed with solutions containing 30 mM potassium, also a stimulant of brain respiration, the response is an immediate pulse of reduction in all cytochromes, followed by a tendency to return to the initial levels. Short trains of pulses of electrical field stimulation result in a biphasic change in the level of reduction of cyt a3, an initial slight reduction being followed by a transient of increased oxidation. This response can be abolished by low-sodium bathing solution but not by ouabain. Cytochromes a, b and c show a simple oxidative response to electrical stimulation; the kinetics of this oxidative response are similar to those of the oxidative transient of the cyt a3 response. Pyridine nucleotides, as measured by their fluorescence, respond to electrical stimulation with a transient oxidation which exhibits slower kinetics than the response of the cytochromes. The high resting levels of reduction of cyt a and cyt a3, the reductive response to ouabain and potassium, and the oxidative response of all cytochromes to electrical stimulation suggest a tighter coupling between oxygen utilization and neuronal function than would be expected if mitochondrial redox states simply reflected changes in phosphate acceptor potential resulting from activity of Na+-K+ ATPase.

Animals↗
PubMed1975

Redox state of respiratory chain enzymes and potassium transport in silkworm mid-gut.

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A.M. and Williams, C.M. (1954) J. Biol. Chem. 209, 915, Shappirio, D.G. and Williams, C.M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A.M. (1957) J. Biol, Chem, 209, 931) have indentified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location. Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc. These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional resporatory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.

Amobarbital↗