Spatial distribution of cardiac transmembrane potentials around an extracellular electrode: dependence on fiber orientation.

Recent theoretical models of cardiac electrical stimulation or defibrillation predict a complex spatial pattern of transmembrane potential (Vm) around a stimulating electrode, resulting from the formation of virtual electrodes of reversed polarity. The pattern of membrane polarization has been attributed to the anisotropic structure of the tissue. To verify such model predictions experimentally, an optical technique using a fluorescent voltage-sensitive dye was used to map the spatial distribution of Vm around a 150-microns-radius extracellular unipolar electrode. An S1-S2 stimulation protocol was used, and vm was measured during an S2 pulse having an intensity equal to 10x the cathodal diastolic threshold of excitation. The recordings were obtained on the endocardial surface of bullfrog atrium in directions parallel and perpendicular to the cardiac fibers. In the longitudinal fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) but only in a region within 445 +/- 112 microns (and 616 +/- 78 microns for anodal pulses) from the center of the electrode (n = 9). Outside this region, vm reversed polarity and reached a local maximum at 922 +/- 136 microns (and 988 +/- 117 microns for anodal pulses) (n = 9). Beyond this point vm decayed to zero over a distance of 1.5-2 mm. In the transverse fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) at all distances from the electrode. The amplitude of the response decreased with distance from the electrode with an exponential decay constant of 343 +/- 110 microns for cathodal pulses and 253 +/- 91 microns for anodal pulses (n = 7). The results were qualitatively similar in both fiber directions when the atrium was bathed in a solution containing ionic channel blockers. A two-dimensional computer model was formulated for the case of highly anisotropic cardiac tissue and qualitatively accounts for nearly all the observed spatial and temporal behavior of vm in the two fiber directions. The relationships between vm and both the "activating function" and extracellular potential gradient are discussed.     

Dendritic and sustained shifts in potential to electrical stimulation of the anuran tectal surface.

1. Recordings of dendritic potentials and sustained potential shifts (SPS) were made from the brain of immobilised frogs during surface tectal electrical stimulation. 2. Single pulses evoked dendritic responses; trains caused decay of dendritic responses on the background of the evoked SPS. 3. The tectal surface SPS declined with distance from the stimulating electrode. 4. The negative surface SPS declined with tectal depth to ca 300 microns, then reversed polarity and increased in amplitude with depth up to 700 microns.     

Comparative study of the space constant of electrotonic decay in the endocardium and epicardium of the rabbit right atrium

The distribution of the electronic potential in the endocardium and epicardium of the rabbit right atrium was studied. The distribution of the electronic potential was studied by the method of intracellular polarization of cardiac fibers via a suction electrode perfused from the inside with a KCl isotonic solution. The space constant of electronic decay along and across cardiac fibers in the endocardium and epicardium of rabbit right atrium was measured. It was shown that the space constant of electronic potential decay in rabbit the right atrium endocardium in the areas of ordered arrangement of trabecules both along (lambda x = 2117 +/- 653 microns) and across (lambda y = 394 +/- 212 microns) the fiber was higher (p < 0.001) than that in epicardium (lambda x and lambda y equal to 1361 +/- 486 microns and 212 +/- 63 microns, respectively). The values lambda x and lambda y do not significantly differ between separate areas of the epicardium. The degree of electrotonic anisotropy in all the structures investigated was almost the same, its value ranging from 2 to 17.     

pH microelectrode: modified Thomas recessed-tip configuration

Through the use of a glass-membrane pH electrode and a water-tight seal a modified Thomas pH microelectrode has been developed. The modified Thomas electrode has a relatively low electrical resistance (10(11) omega), a small sensing chamber (10 microns3), and a rapid response time (10 s) and can be manufactured in both single- and double-barreled configurations. The modified Thomas electrode is designed to measure the intracellular pH of small cells such as those found in the mammalian kidney tubule.     

An ammonia-sensing air gap microelectrode

An ammonia-sensing air gap microelectrode has been designed on the basis of a neutral carrier pH-sensing inner electrode. This electrode has a tip diameter of 2 to 5 microns, has a simple design, is easy to fabricate, and has a long shelf life. Its response to ammonium is linear in the range 3 x 10(-5) to 10(-2) M and its response time (95%) is 10 to 15 s. The electrode was converted to a microsensor for urea by immobilization of urease within its tip. The linear response to urea ranged from 3 x 10(-4) to 10(-2) M and the response time was 15 to 20 s.     

Group conduction velocities and nerve fibre diameters of alpha and gamma-motoneurons from lower sacral nerve roots of the dog and humans.

Single action potentials and their conduction times were recorded extracellularly from dog and human lower sacral nerve roots. Conduction velocity frequency distribution histograms were constructed and peaks of single extrafusal and intrafusal motoneuron distributions were identified. The electrophysiologically measured roots were removed and morphometrically analysed. Nerve fibre diameter frequency distribution histograms were constructed with respect to 3 myelin sheath thickness ranges, and peaks of single motoneuron group distributions were identified. The identified motoneuron classes, characterized by their group peak values of conduction velocity at about 36 degrees C and fibre diameter were: dog: intrafusal: gamma 22(23ms-1/4.8 microns),gamma 21(33/5.7), gamma 1 (43/6.7),gamma beta?(54/10.1) extrafusal: alpha 3(61ms-1/11.7 microns),alpha 2(72/13.6), alpha 11 (81/15.2), alpha 12(86/16.8),alpha 13(95/19) human: intrafusal: gamma 21(15ms-1/5.8 microns), gamma 1(20/6.8), gamma beta?(27/7.2) extrafusal: alpha 3(37ms-1/8.3 microns),alpha 2(50/10.2), alpha 1(60/13.1) The 60 (alpha 3) to 30% (alpha 1-motoneurons) higher conduction velocities in dogs as compared to humans seem to originate in the 40 (alpha 3) to 30% (alpha 1-motoneurons) larger nerve fibre diameters. However, the myelin sheath seemed to be 0.1 to 0.2 microns thinner in dogs than in humans. The pair-values "conduction velocity-fibre diameter" of the alpha and gamma-motoneuron groups were lying on different correlation curves in the velocity-diameter plane indicating structural and/or geometrical differences between alpha and gamma-motoneurons.     

Electrophysiology of the Human Hypophysis

Electrical activity was recorded in the human hypophysis from the neurosecretory fibres of the posterior lobe. In seven patients undergoing trans-sphenoidal hypophysectomy, electrophysiological recording was carried out with a special fine-shielded bipolar concentric electrode with a tip diameter of 30 microns. The rate of unit impulses was modified by anesthetic agents and after intravenous injections of drugs acting at the hypothalamic level.This technique was used to detect the limit between the anterior and posterior lobe in order to perform a selective adeno-hypophysectomy by implantation of yttrium-90 seeds.     

Host-plasmid interactions in Saccharomyces cerevisiae: effect of host ploidy on plasmid stability and copy number

The segregational stability of two chimaeric plasmids has been examined in an isogenic series of haploid, diploid and tetraploid strains of Saccharomyces cerevisiae, constructed by transformation-associated spheroplast fusion. For the highly unstable, ARS-based plasmid YRp7M, a significant increase in its segregational stability was observed with increasing ploidy, while the relatively stable, 2 microns-based plasmid pMA3a showed only a small increase in stability in strains of higher ploidy. The copy number of both pMA3a and the endogenous 2 microns plasmid increased in proportion with the host cell ploidy, while the copy number of TRp7M was increased in the higher ploidy strains but did not correlate with ploidy. These results suggest that the copy numbers of both the 2 microns plasmid and a plasmid derived from it are controlled by a nuclear gene and that, in addition, there are 2 microns sequences, other than those required for the FLP-mediated recombination system, that play a role in maintaining copy number.     

Duodenal microanatomy of the domestic cat (Felis catus)

Duodenal samples were taken from similar locations in six cats, processed, stained, and examined via light microscope. There were no prominent circular folds (plicae circulares) or stratum compactum (lamina subglandularis). The 1072 microns x 201 microns villi were covered by 46 microns high columnar epitheliocytes proximally which decreased in height (41 microns) distally and displayed a 1.1-1.7 microns striated border. Globular leukocytes, mononuclear cells, and twenty-eight goblet cells (exocrinocytus calciformis) per villus were seen. The intestinal gland (crypt of Lieberkuhn) epithelium was 20 microns tall and had a less distinct striated border. The 515 microns simple straight tubular intestinal gland layer displayed distal branching. Many mitotic figures, 12 goblet cells per gland, and occasional columnar to triangular cells with red cytoplasmic granules were seen. The thickness of the lamina propria mucosa (glandular portion) decreased from proximal to distal (563-465 microns). The lamina muscularis mucosa had two layers and decreased in thickness distally (71-28 microns). The proximal muscularis mucosa was penetrated by the ducts of submucosal (Brunner's, duodenal) glands. The tela submucosa decreased in thickness distally (593-192 microns) and contained submucosal glands with 11.5-75 microns lumina for the first 1.5-2.5 cm. However, submucosal glands could be found to a distance of 8 cm. The glandular epithelium ranged from 7.5-22.5 microns in height. Only one type of secretory cell was observed, with both mucous and serous properties. The tunica muscularis ranged from 190-1425 microns (median thickness of 557 microns) and had two layers.     

Yeast 2μm vectors replicate and undergo recombination in Torulaspora delbrueckii

In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 microns sequence yield stable transformants. We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.     

Dye-coupled electrode system for the rapid determination of cell populations in polluted water.

We determined cell populations in polluted waters by using a fuel cell-type electrode. The electrode was constructed from a platinum anode, a silver peroxide cathode, and a membrane filter for retaining microorganisms. The principle of cell number determination is based on sensing a redox dye reduced by the microorganisms with the electrode. Sample solutions containing microorganisms were membrane filtered, and the resulting filter containing microbial cells was attached to the surface of a platinum anode. The electrode was immersed in phosphate buffer solution (0.05 M, pH 7) containing a redox dye (2,4-dichlorophenol-indophenol), and the current generated was measured. The response time of the electrode system was 10 to 20 min, and the current generated was proportional to cell populations above 10(4) cells/ml.     

Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

Electrode System for the Determination of Microbial Populations

Determinations of microbial populations were carried out by using a new electrode system composed of two electrodes. Each electrode was constructed from a platinum anode and a silver peroxide cathode. The anode of the reference electrode was covered with cellulose dialysis membrane. The response time of the electrode system was 15 min in culture broth, and current differences between the two electrodes were proportional to populations of microbial cells in cultures of Saccharomyces cerevisiae and Lactobacillus fermentum. Current differences were reproducible; the average relative error was 5%. Furthermore, cell populations of S. cerevisiae in a fermentor could be continuously estimated by using this electrochemical method.     

Kinetics of follicle growth in the prepubertal gilt.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light and scanning electron microscopy of fallow deer (Dama dama) spermatozoa

No basic differences in size (mean +/- s.d. for at least 300 spermatozoa), shape and ultrastructure of the spermatozoa of fallow deer were detected (1) in comparison to other artiodactyls, (2) between different fallow bucks, and (3) between different months of the fertile season. The total length of the normal spermatozoon was 67.2 +/- 1.2 microns. The flat, paddle-shaped head was 8.2 +/- 0.3 microns long, 4.4 +/- 0.2 microns for the greatest width, 1.9 +/- 0.2 microns for basal width and, approximately 0.7 microns in thickness. The tail measurements were 13.7 +/- 0.3 microns for the midpiece, 0.5 +/- 0.1 microns for the diameter of the midpiece, 42.6 +/- 0.9 microns for the principal piece, and 2.7 +/- 0.6 microns for the endpiece. Spermatozoa with abnormalities such as cytoplasmic remnants and droplets, bent and coiled tails, as well as microcephalic forms were observed.     

Testicular myxosporidiasis in the flat-backed toad, Bufo maculatus (Amphibia: Bufonidae), from Cameroon, Africa.

Macroscopic cysts measuring less than or equal to 860 x 500 microns were found in the testes of a flat-backed toad, Bufo maculatus, collected in Cameroon, West Africa. On histologic examination, the cysts contained numerous spores of a Myxobolus sp. (Myxozoa: Myxobolidae). Spores in fixed tissues measured 9.2 microns long, 8.9 microns wide, and 4.0 microns thick; the range of values for length, width, and thickness were 8.8 to 9.6 microns, 8.6 to 9.4 microns, 3.6 to 4.4 microns, respectively (n = 20). The shape index (length/width) was 1.03, and ranged from 1.00 to 1.09. Pathology was limited to a slight constriction of adjacent seminiferous tubules by the cysts. No host inflammatory response was noted. This myxozoan is distinct from all other members of the genus infecting anurans and is assigned the name Myxobolus bufonis sp. n.     

Existence of respiratory interneurons in the cervical spinal cord of the rabbit

A spinal "respiration" generator has been shown to fire phrenic motoneurones in rhythmic bursts. It is very likely driven through bulbo-spinal inspiratory neurones in intact preparations. Although no direct evidence for respiratory interneurones at the C4-C5 spinal levels has been obtained so far (except for Renshaw cells ), it is currently believed that only few inspiratory inputs to the phrenic motoneurones are transmitted monosynaptically from the medulla. We have tried here to record spinal interneuronal respiratory activities in decorticate, unanaesthetized, vagotomized and curarized rabbit preparations. Different functional categories of interneurones could be identified at the C4-C5 spinal levels: inspiratory and expiratory interneurons with various discharge patterns which rather well correspond to the functional categories of inspiratory and expiratory bulbo-spinal neurones described by Bianchi and Richter. In addition, multiunit inspiratory bursting could be followed over several 100 microns during each electrode penetration. The different categories of interneurones were encountered laterally from 700 to 1,000 microns, at depths ranging from 300 to 500 microns dorsally to the phrenic nucleus, down to the nucleus itself. These results indicate that part of the medullary inspiratory drive is channelled via spinal cord interneurones; they also suggest that an inhibition of phrenic motoneurones from the bulbo-spinal expiratory drive takes place via interneurones.     

Application of L-(+)-Lactate electrode for clinical analysis and monitoring of tissue culture medium

L-(+)-Lactate oxidase (EC 1.1.3.2) was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide. The lactate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. Properties of the enzyme membrane and characteristics of the lactate electrode were clarified for the determination of L-(+)-lactic acid. The lactate electrode responded linearly to L-(+)-lactic acid over the final concentration 0-0.25 mmol/L within 30 s. When the enzyme electrode was applied to the determination of L-(+)-lactic acid in control serum, within-day precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.4% with a mean value of 4.54 mmol/L, 98.0%, and 0.986, respectively. The lactate electrode was sufficiently stable to perform 1040 assays over 13 days operation for the determination of L-(+)-lactic acid. The dried immobilized enzyme membrane retained 84% of its initial activity after storage at 4 degrees C for 12 months. Moreover, the enzyme electrode was applied to the monitoring of culture medium for human melanoma cells. L-(+)-Lactate production and D-glucose consumption were closely related to cell numbers.     

A new system for amplifying 2 μm plasmid copy number in Saccharomyces cerevisiae

The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels. For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature.