Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.     

Action of colchicine on cholinergic and adrenergic mechanisms in guinea pig vas deferens

Guinea pig vas deferens responds to externally applied acetylcholine (ACh) or noradrenaline (NA) by a small rapid contraction (phasi phase) and then a large contraction (tonic phase). The phasic phase was not affected by removal of external Ca²⁺, but tonic phase depended on external Ca²⁺. At lower temperatures the two components became larger and detectable separately. The tonic phase induced by ACh at low temperature (at 20°C) was greatly depressed by brief treatment with colchicine (0.5 μM – 5 μM), although the tonic phase at high temperature (at 37°C) was not affected. Na-induced contraction (phasic or tonic phase) was not changed by the colchicine-treatment. High K⁺ (40 mM)-contracture, which in many cases consisted of a single phase and depended on external Ca²⁺, was also not affected by brief treatment with colchicine. Culture of vas deferens for 3 days in the presence of colchicine, increased the phasic phase of ACh- and NA-induced contractions significantly, but reduced the tonic phase of contractions induced by ACh and NA. Colchicine also reduced high K⁺-contracture, the decrease depending on the period of culture with colchicine. Organ culture with colchicine did not affect the amounts of m-ACh and α-Ad receptors or the IC₅₀ value of ACh and NA on ³H-ligand binding. These results suggest that colchicine specifically interacts with some steps in m-ACh and α-Ad receptor-responsor (e.g. ionophore) coupling without affecting the receptor number or affinity of the receptors for agonists. The mechanisms of action of colchicine are discussed in relation to m-ACh and α-Ad receptor functions.     

A quantitative freeze-etching study on the effects of cytochalasin B, colchicine and concanavalin A on the formation of intercellular contacts in vitro

The effects of concanavalin A (conA), cytochalasin B (CB), and colchicine acting on cell membrane-associated structures, such as surface receptors or submembranal cytoskeletal components, were studied in trypsin-isolated plexus cells reaggregated in vitro. Following reaggregation, these cells form intercellular contacts, i.e. a zonula occludens (ZO) and integrated nexus (gap junctions, GJ). In the control cultures the GJ occupied 4.7%, the tight junctions (TJ) occupied 14 μm/μm² of the surface of the zonula occludens (ZO).All the drugs/lectins studied inhibit contact formation in a characteristic manner. ConA inhibits the formation of GJ (1% of the ZO) and causes 5 times more defective TJ strands than the controls. CB inhibits the assembly of the TJ (75% of the controls) and the GJ (1% of the ZO) when it is administered during the period of contact formation. Colchicine inhibits the formation of GJ (1% of the ZO) and produces 2.5 times more open-end strands of TJ than the controls. All effects are reversible with the exception that integrated GJ are not re-formed after colchicine administration.It is suggested that an intact interaction between surface receptors, intramembranous particles and associated cytoskeletal structures is necessary for remodelling cellular contacts.     

Cell shape changes and transmembrane receptor uncoupling induced by tertiary amine local anesthetics

Tertiary amine local anesthetics (dibucaine, Tetracaine, procaine, etc.) modify cell morphology, concanavalin A (Con A)-mediated agglutinability and redistribution of Con A receptors. Con A agglutination of untransformed mouse 3T3 cells was enhanced at low concentrations of local anesthetics, and the dynamics of fluorescent-Con A indicated that ligand-induced clustering was increased in the presence of the drugs. In contast, these drugs inhibited Con A-induced receptor capping on mouse spleen cells. These effects can be duplicated by combinations of vinblastine (or colchicine) and cytochalasin B suggesting that local anesthetics act on microtubule cell surface receptor mobility and distribution. It is proposed that tertiary amine local anesthetics displace plasma membrane-bond Ca²⁺, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca²⁺ concentration to levels which disrupt microtubular organization. The possible involvement of cellular Ca²⁺ in cytoskeletal destruction by local anesthetics was investigated utilizing Ca²⁺-specific ionophores A23187 and X537A. In media containing Ca²⁺ and cytochalasin B these ionophores caused effects similar to tertiary amine local anesthetics.     

Calcium ionophores A23187 and X537A affect cell agglutination by lectins and capping of lymphocyte surface immunoglobulins

The microtubule-disruptive drugs colchicine and vinblastine alter ligand-induced redistribution of cell surface immunoglobulins and lectin receptors. These effects can be duplicated by treatment of cells with the divalent cation ionophores A23187 and X537A. Ionophore activity was dependent upon the presence of Ca²⁺ (1.8·10^?3?4·10^?4 M) in the culture medium. The K⁺-selective ionophore valinomycin had no effect on ligand-induced redistribution of surface receptors. It is suggested that A23187 and X537A impair membrane-associated microtubules involved in transmembrane control of receptor mobility and topography. In contrast to the action of colchicine and vinblastine that bind directly to microtubules, it is proposed that ionophores indirectly affect microtubules by raising the concentration of Ca²⁺ in the cytoplasm to levels that favor microtubule depolymerization and inhibit microtubule assembly.     

Adhesion of human skin fibroblasts : Effect of tetravalent, divalent and monovalent concanavalin A

Concanavalin A (ConA) pretreatment inhibited the adhesion of fibroblasts to plastic surface in a dose-dependent manner. The ConA effect was reversible and could be inhibited by α-methylmannoside. Pretreatment with cytochalasin B (CB) and colchicine increased the ConA effect. Divalent and monovalent ConA derivatives had no effect on the fibroblast adhesion. This indicates that ligand attachment to ConA receptors is not sufficient to prevent cell adhesion. The requirement of tetravalent ConA for inhibition of cell adhesion suggests that the decrease of lateral mobility of membrane components, which seems to be specific for tetravalent ConA, is responsible for the inhibition of cell adhesion. The enhancement of the ConA effect by colchicine and CB pretreatment suggests an involvement of microtubules and microfilaments in the mobility of ConA receptors of fibroblasts.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

Dopamine receptor-mediated regulation of striatal cholinergic activity: positron emission tomography studies with norchloro[18F]fluoroepibatidine

Large numbers of in vitro studies and microdialysis studies suggest that dopaminergic regulation of striatal acetylcholine (ACh) output is via inhibitory dopamine D2 receptors and stimulatory dopamine D1 receptors. Questions remain as to the relative predominance of dopamine D2 versus D1 receptor modulation of striatal ACh output under physiological conditions. Using positron emission tomography, we first demonstrate that norchloro[18F]fluoroepibatidine ([18F]NFEP), a selective nicotinic ACh receptor (nAChR) ligand, was sensitive to changes of striatal ACh concentration. We then examined the effect of quinpirole (D2 agonist), raclopride (D2 antagonist), SKF38393 (D1 agonist), and SCH23390 (D1 antagonist) on striatal binding of [18F]NFEP in the baboon. Pretreatment with quinpirole increased the striatum (ST) to cerebellum (CB) ratio by 26+/-6%, whereas pretreatment with raclopride decreased the ST/CB ratio by 22+/-2%. The ratio of the distribution volume of [18F]NFEP in striatum to that in cerebellum, which corresponds to (Bmax/K(D)) + 1 (index for nAChR availability), also showed a significant increase (29 and 20%; n = 2) and decrease (20+/-3%; n = 3) after pretreatment with quinpirole and raclopride, respectively. However, both the D1 agonist and antagonist had no significant effect. This suggests that under physiological conditions the predominant influence of endogenous dopamine on striatal ACh output is dopamine D2, not D1, receptor-mediated.     

Acetylcholine Receptors : Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity

Using ¹²⁵iodine-labeled α-bungarotoxin (α-BGT-¹²⁵I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm² at 14 days, subsequently decreasing to 529 receptors/µm² at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-¹²⁵I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.     

Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine

Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for ～12 days in normoxia (Nox; 20% O₂), chronic hypoxia (CHox; 6% O₂), or chronic nicotine (CNic; 10 µM nicotine, 20% O₂), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (～15-min) exposure to hypoxia (5% O₂; ～6× basal), high extracellular K⁺ (30 mM; ～10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ～3× basal), and nicotine (100 µM; ～5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (～9× control), the stimulatory effect of high K⁺ (3–4× basal) and acute hypoxia (～2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (～9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (～2× basal), nicotine, nomifensine, and high K⁺ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Cellular proliferation in the anterior pituitary of the rat during the postnatal period

Summary Cellular proliferation in the anterior pituitary of 2-, 8-, 15- and 30-day-old rats was examined by injection of bromodeoxyuridine 1 h before autopsy. Bromodeoxyuridine incorporated into DNA was detected immunohistochemically by use of a monoclonal antibody. The highest rate of cell proliferation was found in 2-day-old animals; it decreased thereafter during the postnatal period. Possible toxic effects of colchicine on cellular proliferation were examined. Colchicine treatment (10 mg/kg in 8- and 30-day-old animals) significantly decreased the number of bromodeoxyuridine-labelled cells/mm² in 8-day-old rats. Some sections were doubly immunostained for bromodeoxyuridine and various pituitary hormones. The proportion of doubly-immunostained cells to all proliferating cells was generally low, ranging from 23% at 2 days to 32% at 30 days of age.On leave from the Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Salamanca, Spain     

黑眶蟾蜍卵母细胞体外成熟过程中生发泡迁移的研究(英文)

生发泡迁移（ＧＶＭ）是大多数两栖类动物中卵母细胞成熟之前都可以观察到的、涉及细胞核行为的现象。本实验在光镜水平上对激素诱导下的黑眶蟾蜍卵母细胞的ＧＶＭ现象、以及细胞骨架解聚剂类药物———秋水仙素、细胞松弛Ｂ（ＣＢ）对这种激素诱导作用的影响进行了研究。同时,采用ＡＺＡＮ染色法观察了ＧＶＭ过程中生发泡周边纤维骨架的结构变化。将取自刚脱离冬眠期雌体的卵母细胞按不同的培养液、分三个实验组,体外培养不同的时间后,固定、染色、观察。对照组培养液成分为Ｒｉｎｇｅｒ液中加入人绒毛膜促性腺激素和脑垂体;实验组分别增加秋水仙素或ＣＢ。Ｔａｂ．１和ＰｌａｔｅＩ１,４,５,６,７,８,９表明：经过体外培养４ｈ,各组生发泡均向动物极表面发生了迁移。但是,秋水仙素的作用在培养的前２ｈ,对ＧＶＭ表现为促进效应（ＰｌａｔｅＩ５）;而培养的后２ｈ,却表现为抑制（ＰｌａｔｅＩ８）;ＣＢ的作用始终是抑制（ＰｌａｔｅＩ６＆９）。６ｈ后,各组生发泡均告破裂（ＰｌａｔｅＩ１０,１１,１２）。正常情况下,生发泡周围被一环形纤维包围,其外侧有两个纤维化小体（ＰｌａｔｅＩ２）。发育较快者,纤维化小体消失,植物极附近纤维逐渐加厚（ＰｌａｔｅＩ２     

Estrogen target cells. Establishment of a cell line derived from the rat pituitary tumor MtT/F4.

Pretreatment of rabbit kidney cells with cytochalasins B and D (CB, CD) enhanced herpes simplex virus type 2 (HSV-2) DNA infectivity 3- to 6-fold over values obtained using the standard CaCl₂ technique. Cells were pretreated with CB for 4–6 h to achieve infectivity enhancement. A lower concentration of CD, and shorter pretreatment periods, resulted in comparable DNA infectivity. Separate exposure of cells to colchicine, colcemid, or vinblastine increased DNA infectivity 7-, 6-, and 5-fold, respectively, over control values. Additional enhancement was obtained when CD was used together with any one of the aforementioned drugs. Maximal enhancement of HSV-2 DNA infectivity was obtained by pretreating recipient cells with a drug mixture containing colchicine, colcemid, and CD. This treatment maximized infectivity levels 20- to 30-fold over CaCl₂ control values.     

Electron microscopic analysis of the modulation of lymphocyte receptor mobility

The topographic distribution of ferritin-labelled concanavalin A (FT-ConA) bound to the surface membrane of mouse lymphocytes has been analysed by examining ultrathin sections and ghost membranes in the electron microscope. Binding of FT-ConA to lymphocytes at 37 °C did not induce redistribution of ConA receptors at any dose of the labelled lectin. In contrast, in cells treated with colchicine, FT-ConA induced both patch and cap formation. These findings suggest that the modulation of receptor mobility by colchicine occurs at the level of individual receptors. Ultrastructural observations revealed both microtubules and microfilaments in lymphocytes consistent with the suggestion that these cellular structures may be involved in the control of receptor mobility. Analyses using freeze-fracture methods indicated that the distribution of intramembranous particles is not correlated with either the movement of surface receptors or the modulation events. An hypothesis on the modulation of surface receptors by cytoplasmic structures is proposed on the basis of these and previous observations.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.