Enzymatic Isolation Research Articles

Improved enzymatic isolation of fibroblasts for the engineering of autologous skin substitutes

Improved enzymatic isolation of fibroblasts for the engineering of autologous skin substitutes

Applications and benefits of a non-ionic surfactant and artificial oxygen carriers for enhancing post-thaw recovery of plant cells from cryopreservation.

Embryogenic (totipotent) cells cultured in suspension that are capable of regenerating into intact fertile plants, are a routine source for the enzymatic isolation of plant protoplasts (wall-less cells) that are exploited in genetic manipulation studies, particularly for cereals, including rice (Kinoshita and Mori, 2001). Such suspensions are also an alternative source to immature zygotic embryos for transgenic plant production by biolistics (van Schaik et al., 2000). However, the establishment and maintenance of embryogenic suspensions is technically difficult, since for example, morphogenic competence declines progressively with culture at physiologically normal temperatures (Pradhan et al., 1998). Cryopreservation is exploited for the stable, long-term storage of biological tissues at ultra-low temperatures (Moukadiri et al., 2002), negating the requirement to re-initiate and characterize new cell lines to provide a constant supply of competent cells (Lynch et al., 1994). The recovery of frozen cells depends upon prefreeze, cryogenic and post-freeze conditions. However, the transition of cells between ultra-low and physiologically normal temperatures can induce respiratory imbalances, leading to the production of toxic oxygen radicals (Cella et al., 1982; Benson et al., 1992, 1995).

Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice.

The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.

Characterization of stretch-activated cation current in coronary smooth muscle cells.

Stretch-activated ion currents were recorded from vascular smooth muscle (VSM) after enzymatic isolation of single cells from porcine coronary arterioles. Patch pipettes were used to record whole cell current and control cell length. Under voltage clamp in physiological saline solution, an inward cation current (I(CAT)) was activated by 105--135% longitudinal stretch. I(CAT) coincided with an increase in intracellular Ca(2+) concentration. Under current clamp, membrane depolarization was induced by stretch. The magnitude of I(CAT) varied from -0.8 to -6.9 pA/pF at a holding potential of -60 mV. I(CAT) was graded with stretch, inactivated on release, and could be repeatedly induced. A potassium current (I(K)) activated in unstretched cells by depolarization was also enhanced by stretch. In Ca(2+)-free bath solution, stretch-induced enhancement of I(K) was blocked, but I(CAT) was still present. Hexamethyleneamiloride (50 microM), a reputed inhibitor of mechanosensitive channels, blocked I(CAT) and the stretch-induced increase in I(K) but not basal I(K). Grammostolla spatulata venom (1:100,000) blocked basal I(K), blocked stretch-induced increases in I(K), and blocked I(CAT). Iberiotoxin, a specific Ca(2+)-activated K(+) channel blocker, did not alter I(CAT) but blocked the stretch-induced increase in I(K) and increased the magnitude of stretch-induced depolarization. We concluded that longitudinal stretch directly activates a cation current and secondarily activates a Ca(2+)-activated K(+) current in isolated coronary myocytes. Although these two currents would partially counteract each other, the predominance of I(CAT) at physiological potentials is likely to explain the depolarization and contraction observed in intact coronary VSM during pressure elevation.

Electrophysiology of the sodium-potassium-ATPase in cardiac cells.

Like several other ion transporters, the Na(+)-K(+) pump of animal cells is electrogenic. The pump generates the pump current I(p). Under physiological conditions, I(p) is an outward current. It can be measured by electrophysiological methods. These methods permit the study of characteristics of the Na(+)-K(+) pump in its physiological environment, i.e., in the cell membrane. The cell membrane, across which a potential gradient exists, separates the cytosol and extracellular medium, which have distinctly different ionic compositions. The introduction of the patch-clamp techniques and the enzymatic isolation of cells have facilitated the investigation of I(p) in single cardiac myocytes. This review summarizes and discusses the results obtained from I(p) measurements in isolated cardiac cells. These results offer new exciting insights into the voltage and ionic dependence of the Na(+)-K(+) pump activity, its effect on membrane potential, and its modulation by hormones, transmitters, and drugs. They are fundamental for our current understanding of Na(+)-K(+) pumping in electrically excitable cells.

Enzymatic isolation and structural characterisation of polymeric suberin of cork from Quercus suber L.

An enzymatic method has been used to isolate, for the first time, polymeric suberin from the bark of Quercus suber L. or cork. This was achieved by solvent extraction (dichloromethane, ethanol and water), followed by a step-by-step enzymatic treatment with cellulase, hemicellulase and pectinase, and a final extraction with dioxane/water. The progress of suberin isolation was monitored by Fourier transform infrared spectroscopy using a photoacoustic cell (FTIR-PAS). The material obtained (polymeric suberin (PS)) was characterised by solid-state and liquid-state nuclear magnetic resonance, FTIR-PAS and vapour pressure osmometry, and compared with the suberin fraction obtained by alkaline depolymerisation (depolymerised suberin (DS)). The results showed that PS is an aliphatic polyester of saturated and unsaturated fatty acids, with an average molecular weight ( M w) of 2050 g mol −1. Although this fraction represents only 10% of the whole suberin of cork, its polymeric nature gives valuable information about the native form of the polymer. DS was found to have an average M w of 750 g mol −1 and to comprise a significant amount of acidic and alcoholic short aliphatic chains.

Polyembryony in species of the genus Allium

Polyembryony has been detected in 26 Allium species during a series of germination tests. Additionally to those species already known for their polyembryonic tendency, high rates (up to 32%) of twin seedlings have been found in A. splendens and some other species. Genotype effects were obvious between several accessions within the species A. tuberosum. In A. tuberosum, enzymatic embryo sac isolation has been used for demonstration of polyembryonic development in early stages. Genotype and ploidy dependence of the polyembryonic process and ploidy alterations in plants derived from polyembryony are discussed.

Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers

Chemical agents (DTT, EDTA) and enzymes (collagenase, DNAse) are often used for the generation of a single cell suspension from tissue samples. In this study, flow cytometry was used to examine the effect of chemical agents and enzymes on the expression of cell membrane markers of T lymphocytes from tonsils and peripheral blood. Expression of CD4, CD8, CD25, CD38, L-selectin, CD44, αEβ7 and α4β7 were studied. Incubation of lymphocytes with DTT and EDTA resulted in a decrease of CD38, αEβ7 and α4β7 expression. Incubation with collagenase A and DNAse resulted in a decrease of CD25, L-selectin, αEβ7 and α4β7. The results of this study indicate that a careful interpretation is necessary for phenotypic descriptions of lymphocyte populations obtained by enzymatic isolation techniques.

Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function.

Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.

Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius.

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.

The recovery of protein hydrolysate during enzymatic isolation of chitin from shrimp Crangon crangon processing discards

Shell waste from shrimp Crangon crangon processing is a good source of chitin and proteins, contained on a dry basis of the offals in amounts 17.8% and 40.6%, respectively. The digestion of the shells with proteolytic enzymes allow to recovery of the chitin and nutritionally valuable protein hydrolysate. These products were prepared from the shells preliminarily demineralized with 10% HCl solution at 20°C for 30 min using commercially available Alcalase at 55°C and pH 8.5. Recovered protein hydrolysate contained, on a dry basis, 64.3% of protein (N×6.25), 6.24% lipids and 23.4% of sodium chloride and had, at pH 4.0, a minimum solubility, and 81.7% of total nitrogen in the product. The PER value of the obtained product was 2.99 as compared with that for hydrolysates from capelin (2.64) and beef longissimus dorsi muscle proteins (2.81). The charcoal decolorization of the product decreased the PER and amino acid index (EAA) values from 2.99 and 125.4 up to 2.74 and 123.2, respectively. The total amount of residual small peptides and amino acids directly attached to chitin molecules and resistant to enzymatic hydrolysis depends on degree of hydrolysis (DH) and was about 4.4% at DH value of 30%. Such purity of chitin is sufficient for many purposes.

Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.

Mild enzymatic isolation of mannan and glucan from yeast Saccharomyces cerevisiae

Mild enzymatic isolation of mannan and glucan from yeast Saccharomyces cerevisiae

Mild enzymatic isolation of mannan and glucan from yeastSaccharomyces cerevisiae

An enzymatic procedure to isolate polysaccharides from the yeast saccharomyces cerevisiae was developed which would both be environmentally friendly and preserve the native structure of the polymers. Two proteases and three enzyme cocktails were employed. To isolate the cell wall polysaccharides, the cell walls were first separated from the internal components by mechanical cell disruption. The quality of cell disruption was investigated for four types of glass beads with diameters from 0.25 – 1.5 mm and monitored via the inner cellular proteins released. With large glass beads (O = 0.75 – 1.5 mm) both brewer's and baker's yeast cells exhibited more than 90% cell disruption after as little as 12 min, whereas disruption was still incomplete with smaller beads. The isolated cell wall material was purified on microporous membranes, freeze-dried and then incubated with enzymes. Proteases caused protein lysis in the outer cell wall and released soluble mannan. The insoluble glucan was separated by centrifugation and the mannan was purified by dialysis or chromatography. In the digestion with proteases, complete conversion was achieved at concentrations higher than 240 mg g–1 of cell wall material within 26 h. Cell walls from baker's yeast had to undergo a preliminary extraction with petroleum ether. Complete conversion was only possible with one of the enzyme cocktails investigated, and this was achieved after as little as 6 h for cell walls from both baker's and brewer's yeast. The activity of the two other enzyme cocktails investigated was weaker, with brewer's yeast cell walls generally reacting more readily than those of baker's yeast. ZUSAMMENFASSUNG: Im Hinblick auf die Entwicklung umweltfreundlicher Verfahren und den Schutz der nativen Polymerstruktur wurde ein enzymatisches Verfahren zur Isolierung von Polysacchariden aus der Hefe Saccharomyces cerevisiae entwickelt. Eingesetzt wurden zwei Proteasen sowie drei Enzymcocktails. Zur enzymatischen Gewinnung der Zellwand-Polysaccharide wurden zunachst die Zellwande durch einen mechanischen Zellaufschlus vom Zellinneren getrennt. Die Aufschlusgute wurde fur vier Glasperlensorten mit Durchmessern von 0,25 – 1,5 mm untersucht und anhand der freigesetzten innerzellularen Proteine kontrolliert. Sowohl fur Brauereihefezellen als auch Backerhefezellen zeigte sich mit grosen Glasperlen (O = 0,75 – 1,5 mm) bereits nach 12 min ein mehr als 90proz. Zellaufschlus, wahrend bei kleineren Glasperlen der Aufschlus noch unvollstandig war. Das gewonnene Zellwandmaterial wurde an mikroporosen Membranen gewaschen, gefriergetrocknet und anschliesend mit Enzymen umgesetzt. Proteasen zerstorten in der auseren Zellwand die Proteinmatrix und setzten losliches Mannan frei. Das unlosliche Glucan wurde durch Zentrifugieren abgetrennt und das Mannan durch Dialyse oder Chromatographie gereinigt. Bei Umsetzung mit Proteasen war vollstandiger Umsatz ab einer Konzentration von 240 mg g–1 Zellwandmaterial in 26 h moglich. Zellwande aus Backerhefe musten zuvor mit Petrolether extrahiert werden. Von den untersuchten Enzymcocktails war nur mit einem ein vollstandiger Umsatz moglich, der aber sowohl fur Backerhefezellwande als auch fur Brauereihefezellwande schon nach 6 h erreicht wurde. Die Aktivitat der beiden anderen untersuchten Enzymcocktails war schwacher, wobei sich Brauereihefezellwande generell besser umsetzen liesen als Backerhefezellwande.

Cryopreserved porcine hepatocyte cultures

Cryopreservation of freshly isolated hepatocytes is regarded the standard technique for long term storage of liver cells. Frankly, we were not successful in reproducing viability rates of about 70% of that which have been reported by most authors as results of various freezing protocols for hepatocyte suspensions. In fact, we saw mostly devastating results. We assume that intracellular ice crystal formation as well as osmotic changes during freezing and thawing of liver cells cause hazardous effects, especially on membranes of cells after enzymatic isolation, and, thus, generally result in a severe loss in number and impaired specific hepatocyte functions in subsequent culture. We tried to improve results by freezing cell cultures instead. We allowed hepatocytes to regain a more stable condition prior to storage and placed them in tissue flasks in a uniform configuration, rather than to pack cell suspensions in vials or bags under rather indefinable conditions. Porcine hepatocytes (viability 92±2%) were isolated from slaughterhouse organs and cultured in a double gel (sandwich) configuration. At day 3, cultures were rate controlled frozen (RCF) and stored in a cell bank for three hours (Group A) or 14 days at −80°C (Group B), respectively. Non-frozen cells (NF) and cultures subjected to a linear freezing rate of −10°C/min (LFR, Group C) with 3 h of storage served as controls from identical cell batches. Upon thawing, at day 2 of subsequent culture, fluorescence microscopy studies revealed a survival rate of 75±10% (mean±S.D.) in the RCF groups. Time of storage (3 h, 14 d) did not influence results. Survival in Group C was 10±5%. Cell integrity was measured by LDH-release, which indicated a larger damage of cells in the LFR group, and thereby resembled the morphological findings. Functional parameters, such as albumin synthesis and CYT P 450-activity were comparable to non-frozen liver cells at 48 h after thawing in the RCF groups (A+B), and showed significantly higher levels in these groups as compared to the LFR Group (C). We recommend to freeze hepatocytes in culture in a rate controlled fashion rather than cell suspensions. This way a cell bank of cryopreserved hepatocyte cultures for batch controlled investigations can be easily obtained. This could ameliorate the availability of rare (human) cell material and might also improve the quality of data generated from in vitro experiments in hepatology or pharmacology/toxicology with liver cells from identical sources. It remains to be seen whether this technique might also be of value in hybrid bioartificial liver devices to make these systems become readily available upon clinical demand.

Determination of Epidermal Transpiration in Four Cultivars of Nicotiana Tabacum L. Using Epidermal Strips in a Quasi-Steady State System

A quasi-steady state method is presented for quantifying epidermal transpiration of epidermal strips where simple relations between transmembrane fluxes and parameters of diffusibility of penetrating compounds hold. Contrary to most permeability studies, we did not use astomatous, enzymatically isolated, or dried cuticular membranes, because these procedures are largely responsible for the problems cited in the literature. Instead, we used freshly harvested stomatous epidermal strips, thus avoiding the sorption of lipids by the cuticular membranes during enzymatic isolation. Our approach allowed estimation of amounts and composition of intracuticular soluble lipids. Diffusion coefficients (D-values) were calculated with smaller associated standard deviations and an order of magnitude lower than previously reported; the fresh material sorption of the diffusing compound by the membrane and hydration of the cuticular pores was greatly reduced. In the present study the hold-up time (te) ranged from 66.2 ± 0.3 to 110.3 ± 0.9sec. Furthermore, 0.1 μm thick membranes were used, contrary to previous studies of water permeability that used cuticles more than 2 μm thick. Because a small but constant flow of penetrant could be detected during the first half of the steady flow to te, small holes probably did not influence the reported permeability. Permeability coefficients (Pd) in the order of 0.65 × 10−9 ms−1 were calculated. Pd values in the order of 5.68 × 10−3 ms−1 were calculated when incomplete stomatal closure occurred, while when areas of mass flow were detected, Pd values in the order of 1.26 × 10−2 ms−1 were calculated. The degree of contamination of the epidermal strips by cellular debris was quantified and expressed as the total chlorphyll content per exposed surface area of the epidermal strip, and an average of 8.7% contamination was observed compared to the total leaf chlorophyll content. Leakage from the system was calculated to be approximately 0.18 × 10−10 ms−1, which represents an average 2.7% experimental variability. These results are discussed in terms of the limitations associated with using composite membranes that are stomatous and have trichomes, for possible application in drought tolerance selection.

Enzymatic isolation and culture of cement secreting cells from cypris larvae of the barnacle Megabalanus rosa

Isolation of viable secretory cells from cyprid cement glands of the barnacle Megabalanus rosa was achieved by trypsin digestion followed by mechanical treatment. Isolated cells, which were classified into two groups based on cell and granule size, were comparable to earlier descriptions of α‐ and ß‐cells in the cement glands of Balanus balanoides. "α‐Cells”; were large, elongated and contained many small secretory granules (α‐granules), while “ß cells”; were small and contained several larger granules (ß‐granules). α‐Granules expanded significantly when exposed to solutions containing low NaCI concentrations after methanol fixation, whilst ß‐granules did not, suggesting differences in the properties of the respective granules. Isolated cells were cultured for up to one week. The potential application of in vitro cell culture for physiological studies is presented.

Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26.1 +/- 1.5 and 26.1 +/- 1.3 nC microF-1, mid-point voltage of -35.1 +/- 5.0 and -42.2 +/- 1.2 mV and steepness of 16.7 +/- 2.2 and 17.0 +/- 1.9 mV (means +/- S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 +/- 18 and 115 +/- 8 nM (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 +/- 1.0 microM; n = 7), than in guinea-pigs (2.8 +/- 0.3; n = 4). 4. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 +/- 0.1 and 0.9 +/- 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 +/- 0.08 and 0.81 +/- 0.06% ms-1 in rats and 2.43 +/- 0.25 and 0.88 +/- 0.01% ms-1 in guinea-pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5. These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.