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Biomedical subjects

R J Leonard

Publications and source records attributed to R J Leonard.

At least 37 records · Page 2Linked to original sources
PubMed1997

Biodegradable norethindrone (NET:cholesterol) contraceptive implants: phase II-A: a clinical study in women.

Safety, contraceptive efficacy and biodegradability of subdermal norethindrone (NET)-cholesterol implants (Anuelle) were investigated in 19 healthy, sexually active women. Mean serum NET levels in women with four and five pellets, containing 174 mg and 266.5 mg NET, showed a "burst-effect" of 3.17 and 3.71 ng/ml, respectively, within 24 h post implantation. Subsequently, for the four- and five-pellet groups, respectively, the levels declined from 0.75 to 0.40 ng/ml and 1.05 to 0.61 ng/ml during the months 12-15, from 0.40 to 0.11 ng/ml and 0.61 to 0.25 ng/ml up to month 36. The serum NET levels were undetectable at 36 months and beyond, indicating complete biodegradability of NET pellets. Serum E2 levels remained within normal limits (> 50 pg/ml), whereas serum P indicated anovulatory cycles in 78% of the four-pellet group and 99% of the five-pellet group. FSH and LH levels were subnormal and acyclic. Plasma lipids showed reduced total and LDL cholesterol and triglycerides. HDL cholesterol remained unchanged. Drug-related adverse effects were essentially limited to irregular bleeding. There were no pregnancies in either group.

Adolescent↗
PubMed1997

Treatment of ventricular dysphonia with botulinum toxin.

Ventricular dysphonia, traditionally known as dysphonia plica ventricularis, is a voicing disorder in which the false vocal folds are used as a vibratory source in addition to or instead of the true vocal folds. Traditional treatment of ventricular dysphonia has been voice therapy, which may be slow to produce results if the false fold activity masks an underlying problem of the true folds, is long standing, or has produced hypertrophy of the supraglottic structures. We present seven cases of ventricular dysphonia treated with botulinum toxin injection into the false vocal folds followed by speech therapy. The addition of botulinum toxin to the treatment regimen speeds recovery of normal voicing and allows immediate evaluation of dynamic true vocal fold function by the treating professional.

Anti-Dyskinesia Agents↗
PubMed1996

High-conductance calcium-activated potassium channels; structure, pharmacology, and function.

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K+ channels. They are unique in their dual requirement for depolarization and Ca2+ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, alpha and beta. The alpha subunit is a member of the slo Ca(2+)-activated K+ channel gene family and forms the ion conduction pore. The beta subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against alpha and beta subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.

Amino Acid Sequence↗
PubMed1995

Functional role of the beta subunit of high conductance calcium-activated potassium channels.

Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, alpha and beta. We have examined the functional contribution of the beta subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both alpha and beta subunits were much more sensitive to activation by voltage and calcium than channels composed of the alpha subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the alpha subunit alone were insensitive. Thus, alpha and beta subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of co-assembly might contribute to the functional diversity noted among members of this family of potassium channels.

Animals↗
PubMed1995

Stable expression of human Kv1.3 potassium channels resets the resting membrane potential of cultured mammalian cells.

A cDNA clone encoding the human Kv1.3 potassium channel was stably expressed at high levels in a Chinese hamster ovary (CHO) cell line. The expressed currents strongly resemble those recorded from Xenopus oocytes injected with Kv1.3 cRNA, or from resting human T lymphocytes. One function ascribed to Kv1.3 in T lymphocytes is to stabilize the membrane potential, thereby regulating the Ca2+ signalling critical for mitogenic stimulation. In agreement with this hypothesis, we find that the heterologous expression of human Kv1.3 resets the resting potential of CHO cells, setting it to the threshold of activation of Kv1.3. An inhibitor of Kv1.3, margatoxin, depolarized transfected CHO cells to the potential of non-transfected cells. The combination of a steep voltage-dependence of activation with slow and incomplete inactivation appears to confer the ability of the Kv1.3 channel to 'clamp' the Vm of its host cells within a narrow range.

Animals↗
PubMed1994

Chemical synthesis and structure-function studies of margatoxin, a potent inhibitor of voltage-dependent potassium channel in human T lymphocytes.

The 39 amino acid peptide, margatoxin (MgTX), a potent inhibitor of the voltage-activated potassium channel (Kv 1.3) in human T lymphocytes, was synthesized by a solid phase technique. Formation of the disulfide bridges was rapid at pH 8.2. The final product was purified to homogeneity and was physically and biologically indistinguishable from the toxin prepared biosynthetically. The disulfide bridge pairing was similar to that found previously for the related toxin-charybdotoxin (3): from Cys7 to Cys29, from tested for inhibition of 125I margatoxin binding to voltage-activated potassium channels. The results indicate that the three C-terminal residues of MgTX are important for the efficient toxin binding to Kv1.3.

Amino Acid Sequence↗
PubMed1994

Computer-assisted design of an implantable, intrathoracic artificial lung.

A semiempirical mathematical model of convective oxygen transport is used to design a new, low pressure loss, implantable artificial lung that could be used as a bridge to lung transplantation in patients with advanced respiratory failure. The mass transfer and flow friction relations pertinent to the design of a cross-flow hollow fiber membrane lung are described. The artificial lung is designed to transfer over 200 ml/min of oxygen at blood flow rates up to 5 L/min. A compact design and a blood-side pressure loss of < 15 mm Hg allows the device to be implanted in the left chest without the need for a prosthetic blood pump. Surgical implantation of the artificial lung would require the creation of inflow and outflow anastomoses. Oxygen would be supplied via an external source. Blood properties, operating conditions, and empirically determined mass transfer and flow properties are all specified and input into a computer program that numerically solves the design equations. Computer-generated values for the device frontal area, blood path length, and fiber surface area are thereby obtained. The use of this computer-assisted design minimizes the need for extensive trial-and-error testing of prototype devices. Results from in vitro tests of a prototype implantable lung indicate that the mathematical model we describe is an accurate and useful tool in the design of hollow fiber artificial lungs.

Algorithms↗
PubMed1993

Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels.

A novel peptidyl inhibitor of K+ channels has been purified to homogeneity from venom of the new world scorpion Centruroides margaritatus. The primary structure of this 39-amino-acid peptide, which we term margatoxin (MgTX), was determined by amino acid compositional analysis and peptide sequencing. Margatoxin potently inhibits binding of radiolabeled charybdotoxin (ChTX) to voltage-activated channels in brain synaptic plasma membranes. Like ChTX, MgTX blocks the n-type current of human T-lymphocytes (Kv1.3 channel), but compared to ChTX, is 20-fold more potent (half-block at approximately 50 pM), has a slower dissociation rate, and has no effect on calcium-activated channels. To demonstrate that these characteristics are due solely to the purified toxin, recombinant MgTX was expressed in Escherichia coli as part of a fusion protein. After cleavage and folding, purified recombinant MgTX displayed the same properties as native peptide. Replacement of the COOH-terminal histidine residue of MgTX with asparagine resulted in a peptide with a 10-fold reduction in potency. This was due to a faster apparent dissociation rate, suggesting that the COOH-terminal amino acid may play an important role in the binding of MgTX to the Kv1.3 channel. MgTX displays significant sequence homology with previously identified K+ channel inhibitors (e.g. ChTX, iberiotoxin, noxiustoxin, and kaliotoxin). However, given its potency and unique selectivity, MgTX represents an especially useful tool with which to study the physiologic role of Kv1.3 channels.

Amino Acid Sequence↗
PubMed1993

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels.

Charybdotoxin (ChTX) and iberiotoxin (IbTX) are highly charged peptidyl toxins which exhibit 68% sequence identity and share a similar three-dimensional structure. Despite these structural similarities, IbTX and ChTX differ in their selectivity for two types of potassium channels; large conductance calcium-activated potassium (maxi-K) channels and slowly inactivating voltage-gated (Kv1.3) potassium channels. ChTX blocks with high affinity both maxi-K and Kv1.3 channels, while IbTX blocks the maxi-K but not the voltage-gated channel. To identify regions of the toxins which impart this this selectivity, we have constructed by solid-phase synthesis two chimeric toxins, ChTX1-19IbTX20-37 (Ch-IbTX) and IbTX1-19ChTX20-37 (Ib-ChTX), as well as a truncated peptide, ChTX7-37. These peptides were assayed for their ability to inhibit [125I]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [125I]ChTX binding to plasma membranes from brain (Kv1.3 binding). The ability of the peptides to block the maxi-K channel was determined from recordings of single maxi-K channels incorporated into planar lipid bilayers. Block of Kv1.3 was determined from recordings of whole cell currents in Xenopus oocytes injected with mRNA encoding the cloned Kv1.3 channel. Both chimeric toxins inhibited [125I]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [125I]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Tissue response to bioerodible, subcutaneous drug implants: a possible determinant of drug absorption kinetics.

The fibrous tissue compartments that develop in response to the subcutaneous implantation of bioerodible heat-fused rods of norethindrone and cholesterol (85 and 15%, respectively) were studied by light and electron microscopy at various intervals after implantation to determine whether the biological inflammatory response may play a role in drug absorption. Thirty-five regularly menstruating, sterilized (tubal ligation), healthy females each received four Annuelle rods. The microanatomy of seven of the largest implants (135 mg norethindrone) was studied. A dense fibrous biological compartment was found to surround each rod. By light microscopy no abnormal tissue response was revealed. Scanning and transmission electron microscopy showed that the surfaces of the rods were covered by a cellular matrix of mononuclear cells. The fibrous compartment was composed of a loose cellular bed immediately surrounding the norethindrone rod, a dense fibrous connective tissue envelope containing blood and lymphatic vessels, and an outer fatty connective tissue layer. Transmission electron microscopy confirmed that the cellular tissue immediately surrounding the rods was composed mainly of lipid laden macrophages. Norethindrone levels in tissue capsules at 3 and 10.5 months were 0.05 and 8.4% by weight, respectively. These observations suggest that the local inflammatory response plays a role in the active processing of this delivery system. This picture is qualitatively different from the general view of the fibrous capsule as a simple rate limiting membrane. The effects observed in this study suggest that a more complex, functional biological system develops in response to the subcutaneous introduction of a drug delivery device.

Absorption↗
PubMed1992

Selective blockers of voltage-gated K+ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin.

Charybdotoxin (ChTX), a K+ channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltage-activated, and three Ca(2+)-activated. To discern the mechanism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca(2+)-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.

Calcium↗
PubMed1991

Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.

Amino Acid Sequence↗
PubMed1991

Computerized design of speech prostheses.

The use of computerized techniques to assist in the design of palatal and/or glossal prostheses is described. Patients with oropharyngeal resection and associated speech impairment are candidates for such prostheses. Procedures discussed allow for the design of some features of the prosthesis, such as shape, location, and tests of its effect on certain speech parameters, prior to actual fabrication. Advantages and current limitations of the techniques are also discussed.

Computer Simulation↗
PubMed1991

Reverse pharmacology of the nicotinic acetylcholine receptor. Mapping the local anesthetic binding site.

We have been examining the interaction of a local anesthetic derivative, QX-222, with the ion channel pore of the muscle AChR, using a combination of mutagenesis, oocyte expression, and electrophysiology. Single channel recording, together with macroscopic voltage-jump relaxations, provides a measure of the residence time of the open channel blocker within the pore. We have found systematic changes in the apparent affinity of the open channel for QX-222 following amino acid substitutions in the proposed M2 transmembrane helix of each of the four subunits of the AChR. Assigning the number 1' to the residue at the cytoplasmic end of the M2 helix, positions 2',6',10',14', and 18' are modeled as forming the lining of the pore. Polar to nonpolar substitutions at 6' decrease QX-222 residence time, while the opposite effect is seen at position 10'. Nonpolar to polar substitutions have the converse effect. The distance between the aromatic and quaternary amine moieties of QX-222 corresponds almost exactly to the repeat distance of an alpha helix. This structural feature is common to many local anesthetic drugs. We propose a model for the binding of QX-222 within the ion channel of the AChR that is consistent with these observations.

Amino Acid Sequence↗
PubMed1991

Laryngeal cancer: diagnosis, treatment and speech rehabilitation.

Cancer of the larynx occurs most often in men between 50 and 70 years of age. Cigarette smoking and alcohol are responsible for more than 75 percent of cases. Hoarseness is the most common presenting complaint. If hoarseness persists for more than two weeks, laryngoscopy is indicated. Clinical staging utilizes direct laryngoscopy, esophagoscopy and bronchoscopy to exclude synchronous malignancies. The five-year cure rate, with preservation of voice and glottic function, is as high as 90 percent if the lesions of the vocal cords are found in an early stage. Total laryngectomy is required for more extensive disease. Speech rehabilitation has been revolutionized by tracheoesophageal speech techniques.

Bronchoscopy↗