Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Susceptibility to beta-lactam antibiotics in septicemia isolates from twenty-nine European laboratories

In 1984 the European Study Group on Antibiotic Resistance (ESGAR) consecutively collected gram-negative bacilli and staphylococci blood isolates and performed susceptibility testing with 11 antibiotics using the microdilution method. In all 2,578 isolates were collected: 68% gram-negative bacilli and 32% staphylococci. The MICs of ampicillin and cefazoline for the susceptible gram-negative bacilli were 1–8g/ml; of piperacillin0.5–4; of Sch 34343, cefotaxime, moxalactam, ceftazidime and aztreonam0.5–2g/ml; of cefoxitin, cefuroxime and cefamandole0.5–8g/ml. For susceptible staphylococci the MICs of cefazoline and cefuroxime were0.5–1g/ml, and of cefoxitin, moxalactam, ceftazidime and cefotaxime,0.5–32 g/ml. The resistance levels varied between laboratories and countries, being lower in Northern Europe. In clinical protocols on patients with gram-negative septicemia from whom cefazoline-resistant strains were isolated, cefotaxime was the beta-lactam most commonly used (12%). In protocols on patients with staphylococcal septicemia from whom gentamicin-resistant or cefazoline-resistant strains were isolated, the most commonly used beta-lactam was cloxacillin (6%).     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

Association of Phenotypic Changes in B Cell Lymphocytes and Plasma Viral Load in Human Immunodeficiency Virus-Infected Patients

Many B cell abnormalities have been reported in human immunodeficiency virus (HIV)-infected patients, including changes in the expression of , , and CD22 molecules on the cell surface. Phenotypic changes in these markers on B cells isolated from HIV-seropositive patients with high or low levels of plasma viremia were measured. The phenotypic changes in B cells isolated from such patients were compared with the markers on B cells isolated from HIV-seronegative individuals using three-color flow cytometry. HIV patients showed a reduction in the proportion of mature B cells isolated from peripheral blood mononuclear cells compared with B cells isolated from HIV-seronegative individuals. An increase in the proportion of B cells expressing both and molecules on the cell surface was also seen in association with high-HIV plasma viremia. A low plasma viral load was accompanied by a reduction in the proportion of B cells expressing both and molecules to a level comparable to those seen in HIV-seronegative individuals. HIV-seropositive individuals demonstrated an increase in the proportion of committed B cells, as indicated by an increase in the proportion of B cells expressing molecules. This observation may explain the poor humoral response of HIV seropositive patients to neo-antigens. Our results demonstrate that phenotypic changes indicative of in vivo B cell activation and an increase in immature cells are associated with HIV infection, particularly with a high plasma viral load. Phenotypic changes in B cell markers may correlate with functional deficits of B cells.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.     

Possible involvement of microtubules in platelet-activating factor-induced increases in microvascular permeability in vitro

The blood vessels of the rat small intestine were perfused in vitro with a gelatin-containing physiological salt solution (GPSS). The addition of platelet-activating factor (PAF, 5M), podophyllotoxin (50M), colcemid (50M), or nocodazole (50M) to the GPSS for 5 min caused an increase in vascular permeability. This was manifested as an increased trapping of circulating colloidal carbon (CC) within the walls and was assessed using semiautomated image analysis. Pretreatment for 10 min with taxol (5M) in the perfusate significantly reduced the permeability-enhancing effects of all four agonists. Since podophyllotoxin, colcemid, and nocodazole are all microtubule-disrupting agents, and since taxol is a microtubule-stabilizing agent, these results suggest that microtubules are involved in the response of the microvessels to PAF. An explanation based on tensegrity or force-counterbalance is put forward to account for these findings.     

Beeinflussung des post-asphyktischen Wiederaufbaues der Adeninnucleotide in Kaninchenherzenin vivo durch Substratangebot

Zusammenfassung Die Möglichkeit einer Beschleunigung der langsamen postanaeroben Erholung im Status der myokardialen Adeninnucleotide durch ein methodisch einfach durchführbares kontinuierliches Angebot von Substraten, die zum Aufbau von Nucleotiden bedeutungsvoll sein könnten, wurde unterin vivo-Bedingungen am Herzen des Kaninchens anhand von Bestimmungen der Gewebsgehalte von Metaboliten und Substraten des Adenylsäure-Phosphokreatin-Systems und des Glykolysecyclus geprüft. Als anaerobe Belastung diente eine Serie von 4 Asphyxien von 1 mal 3 min und 3 mal 2,5 min Dauer mit zwischenzeitlichen Erholungspausen von 10 min Dauer. Nach Abschluß der raschen Erholungsvorgänge im Herzstoffwechsel wurden die Substrate oder physiologische NaCl-Lösung bis zu einer post-asphyktischen Erholungsdauer von 5 Std in die V. cava sup. oder in das linke Herzohr infundiert. Die Infusion von Bausteinen zurde novo-Synthese des Purinkörpers mit Glycin (6 mol/min), Glutamin (6 mol/min), Asparaginsäure (6 mol/min), Folsäure (2 mol/min), Ameisensäure (0.04 mol/min), Oxalessigsäure (6 mol/min) und Ribose (12 mol/min) und die Infusion von Adenin+Ribose (0.6 bzw. 12 mol/min+12 mol/min) und von Inosin (20 mol/min) resultierte nicht in einem beschleunigten Wiederaufbau der myokardialen Adeninnucleotide; die Ursache wird in einem Mangel an aktivierter Ribose und im Fehlen notwendiger Enzyme im Kaninchenherzmuskel gesehen. Das Angebot von Adenosin (7,5 mol/min) resultierte in einer starken Beschleunigung des post-asphyktischen Wiederaufbaus der Adeninnucleotide und in einer Erhöhung des ATP-Gehaltes und der Summe der Adeninnucleotide um 35 bzw. 39% über die Norm.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Analysis of mutations introduced into the chromosomal immunoglobulin μ gene

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin gene. Homologous recombination between the transferred mutant C region and the wild-type chromosomal C region is expected to introduce the 2-bp deletion into the chromosomal gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal gene deletions.     

Interactions of bradykinin,calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells

Rise in free cytosolic calcium concentrations [Ca²⁺]_i in response to bradykinin and guanosine 5-O-thiotriphosphate (GTPS) was related to the action of phospholipase A₂ (arachidonic acid release). At 900 M extracellular CaCl₂, bradykinin induced a typical Ca²⁺ movement consisting of an initial [Ca²⁺]_i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca²⁺ level at approximately 290 nM. As the extracellular CaCl₂ concentration was reduced to 100 M, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca²⁺ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase [Ca²⁺]_i and saponin treated cells exhibited a very similar pattern of Ca²⁺ mobilization in response to bradykinin. However, with saponin treatment, GTPS (100 M) increased [Ca²⁺]_i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in [Ca²⁺]_i was observed in cells stimulated with both 100 M GTPS and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTPS. Pertussis toxin (PTX) did not affect the bradykinin induced Ca²⁺ mobilization. However, as we showed previously [1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A₂ (PLA₂) activation and Ca²⁺ mobilization but likely by different G subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTPS stimulated release of arachidonic acid appears to be only partially dependent on [Ca²⁺]_i. For example, 10 M ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl₂ concentrations below 300 M while GTPS stimulated a greater release of arachidonic acid at 300 and 100 M CaCl₂ than at 900 M CaCl₂. However, at 100 M CaCl₂, ionomycin increased [Ca²⁺]_i to the same level as bradykinin or GTPS stimulated cells incubated in 900 M CaCl₂.In previously published experiments [1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in [Ca²⁺]_i and occurred in the absence of any added extracellular CaCl₂. TPA did not induce an increase in [Ca²⁺]_i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7.     

Anti-inflammatory and immuno-modulatory effects of novel retinoidlike 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10–23 and the level of plasma fibrinogen (MED 25 moles/kg). When given therapeutically (75 moles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25–75 moles/kg).Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12–15) responded poorly to the T cell mitogen, Con A (2.5 g/ml) and the B cell mitogen, LPS (10 g/ml). The responses were partially restored (30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 moles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.     

Neural nicotinic acetylcholine responses in solitary mammalian retinal ganglion cells

Using the patch-clamp technique,whole-cell recordings from solitary rat retinal ganglion cells in culture have established the nicotinic nature of the acetylcholine responses in these central neurons. Currents produced by acetylcholine (5–20 mol/l) or nicotine (5–20 mol/l) reversed in polarity near –5 mV and were unaffected by atropine (10 mol/l). Agonist-induced currents were blocked by low doses(2–10 mol/l) of the classical ganglionic antagonists hexamethonium and mecamylamine, as well as by d-tubocurarine and dihydro--erythroidine (the latter two do not discriminate clearly between ganglionic and neuromuscular junction receptors). Treatment with the potent neuromuscular blocking agent -bungarotoxin (10 mol/l) did not affect the cholinergic responses of these cells, while toxin F (0.2 mol/l), a neural nicotinic receptor antagonist, readily abolished acetylcholine-induced currents. Thus, the experiments performed to date show that the nicotinic responses of retinal ganglion cells in the central nervous system share the pharmacology of autonomic ganglion cells in the peripheral nervous system. The ionic current carried by the nicotinic channels was selective for cations, similar to that described for nicotinic channels in other tissues. In addition, single-channel currents elicited by acetylcholine were observed in whole-cell recordings with seals > 5 G as well as in occasional outside-out patches of membrane. These acetylcholine-activated events, which had a unitary conductance of 48 pS and a reversal potential of 0 mV, represent the ion channels that mediate the neural nicotinic responses observed in these experiments on retinal ganglion cells.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Bradykinin Antagonizes the Effects of α-Thrombin

-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1M AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 × 10^–6 cm/sec to 4.92 × 10^–6 cm/sec. BK (1M) produced a similar increase to 4.46 × 10^–6 cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1M), prior to addition of AT, reduced the AT permeability coefficient to 2.69 × 10^–6 cm/sec. Addition of NA-BK (1 M) for 5 minutes, then BK (1 M) for 5 minutes, inhibited the effect of BK and of AT (1 M) on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 × 10^–6 cm/sec). AT (1 M) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 M) for 2 minutes also inhibited the effects of -thrombin (1 M) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an -thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 M) with -thrombin (1 M) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators -thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.     

Fast K channels are more sensitive to riluzole than slow K channels in myelinated nerve fibre

The effects of 1–500 M riluzole, a novel psychotropic agent, were studied on the nodal K current of isolated nerve fibres of the frog. When added to the external solution, the substance rapidly and reversibly inhibited slow, fast 1 and fast 2 K components of the tail K current. The concentrations of riluzole inducing half maximum reduction of slow, fast 1 and fast 2 K conductances were 413 M, 24 M and 21 M respectively. It is concluded that the substance is about 20 times more effective in blocking fast than slow K channels.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

Effects of purinergic stimulation on the Ca current in single frog cardiac cells

Ca current (I _Ca) was measured by whole-cell voltage clamp in single cells isolated from frog ventricle, in which the Na current was inhibited by tetrodotoxin (0.3 M) and K currents were blocked by substituting K with 120 mM intracellular and 20 mM extracellular Cs. The influence of stimulation by ATP (0.1–100 M) was assessed in the presence of propranolol (1 M) or pindolol (0.1 M), prazozin (0.1 M) and atropine (10 M). ATP, in the micromolar range, had two types of effect. Like other P₁-purinoagonists, it antagonized the increase in I _Ca elicited by -adrenostimulation. When added alone, 1 M ATP could increase I _Ca up to twofold. An increase in I _Ca was also observed even after it had been maximally enhanced by intracellularly applied cAMP (50 M). Voltage dependence and kinetics of I _Ca were not affected. These effects were considered to be related to P₂-purinoceptor activation. At higher ATP concentrations the increase in I _Ca was less; at 100 M, ATP reduced I _Ca. The ATP-induced increase in I _Ca was prevented by internal perfusion of the cells with GDP [-S] or neomycin, respectively, to block signal transduction to phospholipase C or its phosphodiesterase activity on the polyphosphoinositides. We conclude that P₂purinoceptor stimulation increases the Ca current in frog ventricular cells by a pathway that might involve phosphoinositide turnover.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10^–8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 M) blocked activity evoked by GABA (1–10 M), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 M, and were practically abolished at Ca_i 1 M. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Ca_i reached 1 M.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.