A backward swimming mutant of Chlamydomonas reinhardii

A backward swimming mutant (RL-10) was isolated from Chlamydomonas reinhardii. In contrast to the wild-type flagellum which usually displays a ciliary type beating pattern, the flagella in the RL-10 cells always propagated such undulating waves as found in sperm flagella. This abnormal beating pattern was maintained after the cell was demembranated by a non-ionic detergent (Nonidet P40) and reactivated with ATP. Reactivated axonemes (demembranated flagella) of the wild-type cells changed the beating pattern from the ciliary type to the flagellar type when the Ca²⁺ concentration was increased from 10⁻⁷ to 10⁻⁶ M. However, the RL-10 axonemes did not show such a Ca-dependent change in the beating pattern. Hence the RL-10 flagella might carry defects in the controlling mechanisms of flagellar beating pattern, at sites other than the membrane.     

The effect of serum on lipid synthesis and on the expression of oligodendrocyte marker-enzymes in oligodendrocyte-enriched glial cells in culture

Glial cells were isolated from the cerebra of 7-day old rats and the effect of serum on the development of these cells in culture was studied. The activities of the oligodendrocyte marker-enzymes, 2′3′-cyclic nucleotide 3′-phosphodiesterase and glycerol 3-phosphate dehydrogenase and the synthesis of the myelin-associated sulpholipid, sulphatide, were used to monitor the differentiation of these cells in vitro. The results indicate that serum: (i) represses lipogenesis, cholesterogenesis and sulphatide synthesis, (ii) lowers the expression of 2′3′-cyclic nucleotide 3′ phosphodiesterase and glycerol 3-phosphate dehydrogenase but not of lactate dehydrogenase and (iii) thus impairs the differentiation of oligodendrocytes.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

Studies of in vitro cultivated cells from the smooth muscle organs. 3. Effectiveness of some drugs on pulsation frequency of isolated smooth muscle cells of the chicken amnion]

The effects of some drugs on the beating frequency of isolated cells of the chick amnion cultivated on cover slips were investigated. Cholinergic and adrenergic agonists and antagonists, serotonine, antispasmodics, coronary dilatants and local anesthetics influenced the beating frequency significantly. The isolated chick amnion cells equal in their pharmacological behaviour the intact chick amnion and smooth muscle cells of mammals but differ from isolated beating heart cells.     

Effects of cardioactive drugs on the beating activity of myocardial cell cultures isolated from offspring of trained and untrained pregant rats

Summary Primary cultures of beating myocardial cells were obtained from 5-d-old offspring of trained (T) and untrained (UT), pregnant, Sprague-Dawley rats. The myocardial cells from the T and UT groups were evaluated for their beating responses to three cardioactive drugs: verapamil (V), isoproterenol (ISO), and propranolol (PRO). The myocardial cell cultures from the UT group showed complete loss of beating when the calcium (Ca⁺⁺) antagonist, V, was added to the cultures for 1 h or more; the T group was able to show some beating at comparable concentrations and durations of exposure with V. The beta agonist, ISO, markedly stimulated the beating rate of both the T and UT groups, but the beating rates were higher in the UT group at comparable concentrations and durations of exposure than with the T group. When the cultures were pretreated with the beta blocker, PRO, before treatment with ISO, a concentration inhibitory effect on the beating rate was observed in both groups. However, the T cultures were more sensitive to the inhibitory effects of PRO. These results demonstrate that primary cultures of rat myocardial cells isolated from the offspring of trained and untrained pregnant rats show differential beating responses to three well-known cardioactive drugs. This study was supported by grants from the American Heart Association, Texas Affiliate, and the University of Texas Research Institute.     

A novel purification method of murine embryonic stem cell- and human-induced pluripotent stem cell-derived cardiomyocytes by simple manual dissociation

Cardiomyocytes derived from embryonic stem cells (ES-CMs) and induced pluripotent stem cells (iPS-CMs) are useful for toxicity and pharmacology screening. In the present study, we found that cardiomyocyte-rich beating cell clusters (CCs) emerged from murine embryonic stem cell (mESC)-derived beating EBs and from human-induced pluripotent stem cell (hiPSC)-derived beating EBs dissociated by gentle pipetting with a thin glass pipette. The percentage of cardiac troponin T (cTnT)-positive cells in the beating CCs obtained from mESC-derived and hiPSC-derived beating EBs was higher (81.5% and 91.6%, respectively) than in beating-undissociated EBs (13.7% and 67.1%, respectively). For mESCs, the yield of cTnT-positive cells from beating CCs was estimated to be 1.6 times higher than that of beating EBs. The bromodeoxyuridine labeling index of mouse ES-CMs and human iPS-CMs in beating CCs was 1.5- and 3.2-fold, respectively, greater than those in beating EBs. To investigate the utility of the cells in toxicity assessment, we showed that doxorubicin, a cardiotoxic drug, induced myofilament disruption in cardiomyocytes isolated by this method. This simple method enables preparation of mouse ES-CMs and human iPS-CMs with better proliferative activity than beating EBs not dissociated by pipetting, and the cardiomyocytes are useful for drug-induced myocardial toxicity testing.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

NOA1 is an essential GTPase required for mitochondrial protein synthesis

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1–/– cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1–/– cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.     

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3′-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′-O-β-diglucoside by spectroscopical methods.     

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl₂ (n = 22). Patch-clamp techniques were used to record I_Kr and I_K1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl₂, at concentrations that selectively block I_K1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I_K1 in 53% (20/38) of the cells studied, but presence of I_Kr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I_K1 in the majority of cells, but robust expression of I_Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I_Kr due to the absence of I_K1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.     

Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro

On the basis of cell shape, refractility under phase optics, spontaneous beating and nucleolar number, two cell types have been distinguished in embryonic heart cell cultures: M cells (myoblastlike) and F cells (fibroblast-like). However, by different criteria, more than two cell types have been found in the heart in vivo. In the present study, heart cell types are redefined by properties which can be used for identifying cells both in vitro and in vivo. Trypsin-dissociated cells from 7-day chick hearts were cultured for 24 h. PAS staining indicated that all M (thick, refractile) cells contain glycogen (gly⁺), while most F (spread) cells do not (gly⁻). Under the electronmicroscope, only gly⁺ cells contain myofibrils; most of these cells beat spontaneously in culture. Gly⁻ cells do not beat. Gly⁺ cells with myofibrils as well as gly⁻ cells are found in the inoculum and in the heart in vivo. The ultrastructure of gly⁺ cells in vitro and of muscle cells in the myocardium in vivo is similar. Therefore, it is concluded that gly⁺ cells in culture are derived from the myocardium (muscle) of the heart. Gly⁻ cells are apparently derived from the epicardium, the endocardium and the endothelial lining of blood vessels.     

Detection of heavy metal toxicity using cardiac cell-based biosensor

Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner which offers insight into the physiological effect of an analyte. To investigate the potential applications of cell-based biosensors on heavy metal toxicity detection, a novel biosensor for monitoring electrophysiological activity was developed by light-addressable potentiometric sensor (LAPS). Extracellular field potentials of spontaneously beating cardiomyocytes could be recorded by LAPS in the range of 20 μV to nearly 40 μV with frequency of 0.5–3 Hz. After exposed to different heavy metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Fe³⁺, Cu²⁺, Zn²⁺; in concentration of 10 μM), cardiomyocytes demonstrated characteristic changes in terms of beating frequency, amplitude and duration under the different toxic effects of ions in less than 15 min. This study suggests that, with the physiological monitoring, it is possible to use the cardiac cell-based biosensor to study acute and eventually chronic toxicities induced by heavy metal ions in a long-term and no-invasive way.     

Beating rate of isolated neonatal cardiomyocytes is regulated by the stable microtubule subset

We investigated the roles of microtubule (MT) dynamics (growth and shrinkage), the stable, nongrowing MT subset, the posttranslationally detyrosinated MT subset, and artificially elevated tubulin levels in the negative regulation of heart cell beating rate. We manipulated the MT populations in isolated, neonatal cardiomyocytes obtained from normal animals in several ways and then measured heart cell beating rate directly. We found that the stabilized population of MTs was sufficient to maintain a normal beating rate, whereas MT dynamics and detyrosination made no observable contribution. Furthermore, by directly and acutely increasing the level of tubulin within otherwise normally beating cells, we found that the increased tubulin (and MT) levels further depressed the beating rate. In conclusion, the stabilized MT subset is sufficient to maintain the normal beating rate in these cells, whereas increasing the MT density depresses it.     

Calcium activation of macrocilia in the ctenophoreBeroë

1. Macrocilia on the lips of the ctenophore Bero? are usually quiescent, but can be activated to beat rapidly and continuously by various stimuli. 2. During feeding, macrocilia beat actively and serve to spread the lips of Bero? over its prey. 3. Vigorous, repetitive mechanical stimulation of the lips evokes widespread activation of macrocilia via a pathway that is probably neural. 4. Extracellular electrical stimulation (DC or bipolar pulse-trains) elicits immediate activation of macrocilia on lip pieces, but not on dissociated cells. 5. Macrocilia on lip pieces are activated to beat by high KCl artificial sea water (ASW), but not by high KCl Ca-free ASW. Continuous beating for long periods is also elicited by high Ca ASW or Mg-free ASW, but not by Ca-Mg-free ASW. Addition of La, Cd, Co or Mn (10 mM) to high KCl ASW reversibly blocks activation. Verapamil, D-600, nifedipine, or BAY K 8644 (10 microM) has no effect on KC1-induced activation, but the anticalmodulin drug W-7 (10 microM) reversibly inhibits beating. 6. Mild heat treatment dissociates macrociliary cells from lip tissue. Such isolated macrociliary cells usually beat continuously in normal sea water, and swim in circular paths. Ca-free ASW, or addition of Co or Mn to ASW, inhibits beating of dissociated cells. High KCl ASW activates beating of quiescent, isolated macrociliary cells. 7. Ca-Mg-free ASW inhibits beating of dissociated macrociliary cells, and return to Mg-free ASW activates motility, allowing one to activate macrocilia on isolated cells simply by addition of Ca.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid loss of sensitivity to tetrodotoxin by chick ventricular myocardial cells after separation from the heart

We reported previously that chick myocardial cells placed into monolayer cell culture lost tetrodotoxin (TTX) sensitivity when tested at 72 h. To further characterize the change, ventricular myocardial cells were dispersed from chick embryos 14–16 days old; these hearts are TTX-sensitive before dispersal. Intracellular microelectrode penetrations were made into spontaneously beating cells at 9–72 h after culturing. No TTX-sensitive cells were found. Spontaneous action potentials with concomitant contractions continued in the presence of TTX (8 μg/ml), and the maximum rate of rise of the action potentials (+ _max) (control of 2–20 V/sec) was not reduced. Since the cells did not adhere to the vessel before 9 h, suspensions of cells were studied 1–8 h after dispersal to determine the rapidity of the loss of TTX sensitivity; all cells which contracted spontaneously or responded to electrical stimulation continued to beat in TTX. Addition of cycloheximide or actinomycin D did not prevent the loss of TTX sensitivity. The loss is not due to the use of trypsin (0.01 %) because dispersal by collagenase also resulted in loss of TTX sensitivity. Furthermore, cells separated mechanically (from 8-day-old hearts) also lost TTX sensitivity. In addition, loss of TTX sensitivity did not occur in frog sartorius muscles organ cultured for several days in 0.01 % trypsin. The loss of TTX sensitivity occurred even in multilayered cell cultures. Chronic exposure to carbachol or isoproterenol did not prevent the loss. However, elevation of K⁺ in the medium (12–60 mM) prevented or reversed the loss of TTX sensitivity in some cells (˜50 %), although + _max remained low. Hence, the loss of TTX-sensitive fast Na⁺ channels upon cell dispersal (a) occurs very rapidly (less than 60 min), (b) is not due to the use of trypsin, (c) is independent of protein synthesis, (d) is not solely a function of cell association, (e) is not influenced by neurotransmitters, and (f) is prevented or reversed by culturing in elevated [K⁺]₀. The mechanism of the changes in characteristics of the cation channels remains to be elucidated.     

18-Ethynyl-deoxycorticosterone inhibition of steroid production is different in freshly isolated compared to cultured calf zona glomerulosa cells

The inhibiting effects of 18-ethynyl-deoxycorticosterone (18-E-DOC) as a mechanism-based inhibitor on the late-steps of the aldosterone biosynthetic pathway were examined in calf adrenal zona glomerulosa cells in primary culture and in freshly isolated calf zona glomerulosa cells. 18-E-DOC inhibited the stimulated secretion of aldosterone and 18-hydroxycorticosterone in a similar dose-response and time fashion. No significant differences were found between the inhibition in cultured and freshly isolated cells (K_i of 0.25 vs 0.26 μM) Corticosterone secretion stimulated by ACTH or angiotensin II was also cultured in freshly isolated zona glomerulosa and fasciculata cells, but was not inhibited in cultured calf adrenal cells. Cortisol secretion stimulated by ACTH was not inhibited by 18-E-DOC in cultured zona fasciculata adrenal cells, but was inhibited in freshly isolated zona fasciculata cells with a K_i of 48 μM. The secretion of 18-hydroxyDOC or 19-hydroxyDOC stimulated by ACTH was not inhibited by 18-E-DOC. The bovine adrenal has been reported to have cytochrome P-450 11β-hydroxylases that can perform the various hydroxylations required for the synthesis of cortisol and aldosterone in the different areas of the adrenal. In other species a distinct 11β-hydroxylase which participates in the biosynthesis of aldosterone and is located in the zona glomerulosa has been described. These studies with the mechanism-based inhibitor, 18-E-DOC, suggest that the bovine adrenal functions in a manner very similar to that of other species and raises the possibility that a distinct 11β-hydroxylase with aldosterone synthase activity might be present, but has not been cloned as yet.