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Biomedical subjects

I Jacobson

Publications and source records attributed to I Jacobson.

At least 19 recordsLinked to original sources
PubMed2001

Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells.

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.

Acetylcholine↗
PubMed2001

Chronic oesophagitis in the cat.

BACKGROUND: Our understanding of the pathophysiology of gastro-oesophageal reflux disease (GERD) in man is limited. The aim of the present study was to establish a long-term (>1 year) animal model for reflux oesophagitis which would allow us to study various aspects of the development of chronic reflux oesophagitis. METHODS: Myotomy was carried out in the gastro-oesophageal junction in eight cats; seven other cats were sham-operated. Before the operation, and every 2 months thereafter, oesophagoscopy was carried out, biopsies were taken for histology, and manometry was performed to determine the lower oesophageal sphincter pressure (LESP). The cats were killed 1 year after the operation. RESULTS: The myotomy operation resulted in a significantly decreased LESP. In oesophageal biopsies from these cats, there was a varying degree of oesophagitis starting already 2 months after surgery. In six of the eight myotomized cats there was hyperplasia of the stratum basale, and cardiac type metaplasia was observed in two cats. The control cats showed no significant changes in LESP or in the histology of the oesophagus. CONCLUSIONS: In cats followed for more than a year, myotomy in the gastro-oesophageal junction results in reflux oesophagitis similar to that seen in patients with chronic gastro-oesophageal reflux.

Animals↗
PubMed1999

Management of interferon relapsers.

Relapse in patients who have evidenced an initial response to a course of interferon monotherapy for chronic hepatitis C virus (HCV) infection has been a major frustration. At least 70% of responders will relapse after a 6-month course of treatment and more than 50% will relapse after a 12-month course of treatment, usually within the first 3 to 6 months after treatment discontinuation. At most, half of patients relapsing after an initial 6-month course will have a sustained response to a second, 12-month course of interferon monotherapy. A more promising second-line regimen is the combination of interferon plus ribavirin. A recent multicenter international trial has shown that this regimen results in a sustained virologic response rate of nearly 50%, fully 10-fold higher than the sustained response rate observed with a second 6-month course of interferon alone (49% vs 5%, respectively). In patients with non-1 genotype and viral levels no greater than 2 million copies/mL, the likelihood of sustained response to the second course of combination therapy was 100%. Biochemical and virologic responses (normalization of liver enzymes and disappearance of HCV RNA from the serum) were frequently accompanied by histologic improvement. HCV-RNA levels < or = 2 million copies/mL and non-1 viral genotype predicted treatment response, whereas age, gender, body weight, source of infection, type of interferon used for the first round of treatment, and elapsed time since first treatment had no effect. Based on these data, combination therapy with interferon and ribavirin is now considered the treatment of choice for relapsers. It is not clear whether 12 months of combination therapy might have produced even better rates of viral eradication in relapsed patients.

Antiviral Agents↗
PubMed1999

Lectin histochemistry of the esophagus in several mammalian species.

The mucosa of the esophagus consists of stratified squamous epithelium that has a considerable resistance to injury. Intercellular glycoconjugates appear to constitute a major permeability barrier in the superficial portion of the esophageal mucosa. In the present study, we used a panel of lectins to investigate the differences in glycoconjugate production among different mammalian species. A battery of 12 lectins was used to study binding in sections from the esophagus of 6 mammalian species, including man. In general, the strongest staining was obtained in the stratum superficiale and the weakest staining in the stratum germinativum. In rabbit esophagus, exposure to pepsin/HCl produced a superficial damage to the epithelium, a considerable decrease in electrical resistance and a decreased staining of the esophageal epithelium with selected lectins. Pretreatment of the esophageal mucosa with sucrose octasulfate, a compound with protective properties, prevented, to some extent, the decrease in resistance and lectin staining.

Animals↗
PubMed1998

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Patch clamp detection in capillary electrophoresis.

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.

Animals↗
PubMed1997

Ca2+ ion permeability properties of (R,S) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in isolated interneurons from the olfactory bulb of the rat.

The aim of the study was to investigate the divalent cation permeability of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors expressed in interneurons of the olfactory bulb. Kainic acid (KA) was used as agonist to activate AMPA-receptor-mediated currents, which were recorded with the use of the patch-clamp technique. In interneurons acutely isolated from the olfactory bulb, the current responses to KA showed linear/outwardly rectifying current-voltage (I-V) relationships with a positive average reversal potential of +7 mV in normal external medium (1 mM Ca2+, 1 mM Mg2+). Raising the external Ca2+ concentration to 10 mM suppressed the amplitude, whereas omission of Ca2+ enhanced the amplitude of the current. Spectral analysis of the increase in current variance produced by KA indicated that the decreased amplitude observed in 10 mM Ca2+ was accompanied by a reduction in the apparent single-channel conductance. Raising the concentration of Mg2+ from 1 to 10 mM had a weak depressant effect on the KA-evoked current amplitude. No shift in the reversal potential was observed when the concentration of Ca2+ or Mg2+ was changed from 1 to 10 mM. Increasing the external medium concentration of Ca2+ to 60 mM not only further depressed the amplitudes of the KA-evoked currents but also gave a pronounced leftward shift in the average reversal potential to -32 +/- 9 (SE) mV (N = 7). For neurons in primary culture, current responses to KA also showed linear/outwardly rectifying I-V relationships with a positive average reversal potential in normal external medium. Substituting N-methylglucamine for Na+ and increasing the Ca2+ concentration to 10 mM gave a leftward shift in the average reversal potential from +9 +/- 3 mV to -47 +/- 4 mV (N = 11) and caused a marked reduction in the amplitude of the KA-evoked currents at negative potentials. The permeability properties of the studied AMPA receptors were well predicted by the Eyring rate model (symmetrical, 2 barriers, 1 site). The model gave a pCa2+/pK+ permeability ratio of 0.06 for acutely isolated interneurons and 0.14 for interneurons in primary culture. The constant field theory, which failed to successfully reproduce all the experimental data, gave corresponding low permeability ratios of 0.18 and 0.40 for acutely isolated cells and cells in primary culture, respectively. Thus it is concluded that interneurons in the olfactory bulb mainly express AMPA receptors with low permeability to Ca2+ ions.

Animals↗
PubMed1997

Idiopathic subglottic stenosis: diagnosis and endoscopic laser treatment.

The records of 15 patients with idiopathic subglottic stenosis treated at Royal North Shore Hospital, Sydney, between 1980 and 1994 were reviewed. All were female and had similar characteristic clinical and histopathologic features. Endoscopic laser vaporization was the primary treatment in 12 patients and was successful in maintaining the airway of 8 of these 12; this outcome indicates that the disease can be managed, at least initially, by endoscopic laser treatment in most cases.

Adolescent↗
PubMed1996

Patch-clamp detection of neurotransmitters in capillary electrophoresis.

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Animals↗
PubMed1996

Studies on the identity of the rat optic nerve transmitter.

The possible role of glutamate, aspartate, sulfur-containing excitatory amino acids and gamma-glutamyl peptides as major transmitters in the rat optic nerve was evaluated. Four days following optic nerve lesion the K(+)-evoked Ca(2+)-dependent glutamate release was reduced to 31 +/- 16% (+/- S.D., n = 9) comparing release from slices of the denervated (contralateral to the lesion) and non-denervated (ipsilateral) superior colliculus, indicative of a major transmitter function for glutamate. However, significant decreases in glutamate release could not be detected seven days following the lesion (n = 5). Other studies have shown that optic nerve denervation induce formation of synapses of non-retinal origin and cause other cellular changes which may reduce the effect of deafferentation on glutamate release after 7 days. No significant change was observed in aspartate release following the lesion. The concentrations of cysteine sulfinate, cysteate, homocysteine sulfinate, homocysteate and O-sulfo-serine in the optic layers of the superior colliculus were below 1 nmol/g tissue (n = 6). Theoretical considerations indicate that this level is too low for a function of any of these as a major optic nerve transmitter. All postsynaptic components in the rat superior colliculus response, evoked by electrical optic nerve stimulation, were reduced by kynurenate (1-10 mM), a broad spectrum glutamate-receptor antagonist. The study gives further support for the view that glutamate is a major transmitter in the rat optic nerve.

Amino Acids, Sulfur↗
PubMed1995

Expression of Ca(2+)-ion permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in Xenopus oocytes injected with total RNA from human epileptic temporal lobe.

By using the Xenopus oocyte as an expression system, we have performed a series of experiments in order to examine the divalent cation-permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors from the human epileptic temporal lobe. Xenopus oocytes, injected with total RNA from the epileptic temporal lobe, were tested for expression of receptors by a conventional two electrode voltage-clamp technique. Administration of glutamate and AMPA gave small or no clear current responses, whereas kainate produced large inward non-desensitizing currents. The current responses evoked by kainate were concentration dependent. Experimental data gave a Hill coefficient of 1.06 and an EC50 value of 87 microM. The current to voltage relationship showed an inward rectification and when the concentration of divalent cations were enhanced, there was a shift in the reversal potential from -11 mV (2 mM Ca2+) to 12 mV (60 mM Ba2+). This yielded a pBa2+/pK+ permeability ratio of 1.6 when the constant field equation was used. The amplitude of the currents evoked by 600 microM kainate in solutions containing higher Ba(2+)-ion concentrations was markedly diminished (46% in 10 mM Ba(2+)- and 75% in 60 mM Ba(2+)-solution), when compared to those obtained in normal Ringer's solution, suggesting interactions between different cation species and/or screening of surface charges.

Animals↗
PubMed1995

The effect of anosmia on MK-801-induced behaviour in mice.

Systemic administration of N-methyl-D-aspartate (NMDA) receptor antagonists induces a well defined behaviour in rodents characterized by, for example increased locomotion and ataxia. It is not clear in what brain region(s) NMDA antagonists induce this behaviour. We have studied the possible involvement of olfactory pathways by making adult mice anosmic via intranasal injection of zinc sulphate, a procedure that is known to destroy the olfactory epithelium. The NMDA antagonist MK-801 was given intraperitoneally (0.1-1.0 mg/kg) and the animals were scored for locomotion and ataxia 60-90 min later. Before MK-801 administration, olfactory-lesioned mice did not differ from non-lesioned controls with regard to locomotion or ataxia. MK-801 caused locomotor activation (> or = 0.2 mg/kg) and ataxia (> or = 0.5 mg/kg) in both groups. In general, olfactory-lesioned animals showed more locomotion and less ataxia after MK-801 administration than non-lesioned animals. Lesioned animals displayed 2.0- (P < 0.05) and 3.7-fold (P < 0.05) more extensive locomotor activation than non-lesioned animals after 0.5 and 1.0 mg/kg of MK-801, respectively. No difference in the degree of ataxia was seen between the two groups at 0.5 mg/kg, whereas non-lesioned animals showed a 2.1-fold higher degree of ataxia after 1.0 mg/kg of MK-801, indicating that the enhanced MK-801-induced locomotor activation in olfactory-lesioned mice was not simply due to less ataxia. These results suggest that olfactory input is involved in NMDA antagonist-induced behaviour.

Animals↗
PubMed1995

Pentobarbitone modulation of NMDA receptors in neurones isolated from the rat olfactory brain.

1. The action of pentobarbitone on the N-methyl-D-aspartate (NMDA) receptors of neurones freshly dissociated from the olfactory bulb and olfactory tubercle has been studied using patch-clamp techniques. 2. Pentobarbitone produced a concentration-dependent depression of the currents evoked by NMDA with an IC50 value of c. 250 microM. 3. Analysis of the NMDA-evoked noise produced power spectra that could be fitted by the sum of two Lorentzians with corner frequencies of 17 and 82 Hz. Pentobarbitone increased the corner frequency of the high frequency component but did not alter the apparent single channel conductance estimated from the noise. 4. Single channel recordings in either the cell-attached or outside-out patch configurations revealed that NMDA (20 or 50 microM) opened channels with a main conductance level around 55 pS and a principal subconductance around 44 pS. The uncorrected mean open time of the channels was 3.4 ms and mean burst length was 6.0 ms. Mean cluster length was about 12 ms. 5. Pentobarbitone produced a concentration-dependent reduction in both mean open time and burst length. Mean cluster length was much less affected. Pentobarbitone did not decrease unitary current amplitude or bias the open-state current amplitude distribution in favour of a particular substate. 6. From these data it appears that pentobarbitone depresses the inward current evoked by NMDA by reducing the probability of channel opening and this results from a shortening of the lifetime of the channel open state and by decreasing burst length.

Animals↗