Early and delayed changes in potassium transport during the initiation of cell proliferation in CHO culture

Stimulation by serum of cell proliferation in G1-arrested culture of Chinese hamster ovary cells CHO-K1 was accompanied by an early (during the first minutes) and delayed (2-10 h) activation of Na+,K+-ATPase and an increase in cell K+ content from 0.5-0.6 to 0.7-0.8 mmol per gram protein. Isoproterenol acted synergistically with serum in eliciting both early and delayed changes in K+ transport and in stimulating G1----S transition. Isoproterenol alone (without serum) induced a transient increase in K+ influx via Na+,K+-ATPase without changing the cell K+ content or having any mitogenic effect. Theophylline enhanced the serum-induced early activation of Na+,K+-ATPase but inhibited both the delayed increase in cell K+ and the G1----S transition. Early serum-induced increase in K+ transport was not affected by cycloheximide, whereas net accumulation of cell K+ was abolished by the drug. It is concluded that the early and the delayed activation of Na+,K+-ATPase induced by mitogens can be dissociated; the early ionic response is related to the primary transduction of membrane signal, whereas the delayed modulation of ion transport via Na+,K+-ATPase has another function and is associated with cell growth.     

Elaboration of the method of "quiescent cells" for the study of heliomycin-synthesizing system of Streptomyces olivocinereus

A resting cell procedure was developed for S. olivocinereus. Washed mycelium of S. olivocinereus produced heliomycin for a short incubation period of 1.5 hours in a nitrogen-free medium containing a buffer solution, salts, a source of carbon and an inhibitor of protein synthesis. With the developed procedure production of heliomycin in the system of resting cells was investigated. For this purpose mycelium collected during various phases of S. olivocinereus development in batch cultures was used. It was found that in the batch cultures the rate of heliomycin production by the 24th hour of the development was comparable with that of the antibiotic accumulation in the resting cell system. After that period it markedly decreased by the 48th hour. This deviation in the dynamics of heliomycin production in batch cultures and the resting cell system can serve as a basis for further studies on heliomycin biosynthesis control by the carbon source.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described.     

Early and late changes in potassium transport during the start of proliferation in CHO cell cultures. The action of serum, isoproterenol, propranolol and theophylline

The stimulation of DNA synthesis by serum is accompanied by early (30 minutes) and late (2-8 hours) increase in ouabain-sensitive rubidium (potassium) influx and the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram protein in CHO-K1 cells. Isoproterenol alone induces the transient increase both in potassium influx via Na,K-ATPase and in potassium efflux without any effect on intracellular potassium content and cell proliferation. Isoproterenol acts synergistically with serum in eliciting the early and late changes in potassium transport and in stimulating G1----S transition. The combination of serum and theophylline produces a rapid increase in potassium influx, however, it does not stimulate DNA synthesis and does not induce any later increase in intracellular potassium content. It is concluded that early and late activation of Na,K-ATPase by mitogens can be dissociated; the Na,K-ATPase activation is involved in mitogenic response when producing the sustained potassium influx and the elevation of intracellular potassium content during G1----S transition.     

Frequency modulation of theta bursts of the septal neurons in the removal of ascending afferent effects in rabbits

Medial septal-diagonal band (MS-DB) units were examined extracellularly in chronic rabbits under two experimental conditions: 1) in an intact septum, under anaesthetic doses of pentobarbital (40 mg/kg, i.v.); 2) in a basally undercut septum of unanaesthetized rabbits. The background rhythmic burst activity was undistinguishable in both states. Low-frequency electric stimulation of afferent inputs (MFB, CA1, LS) led to entrainment of the theta-cycles. The upper limit of following was almost normal in the undercut septum, but was strongly reduced under pentobarbital. In units with the driving "by pause", the duration of the initial silent period under pentobarbital was increased almost twofold but in the basally undercut septum it was the same as in the normal state. Some MS-DB units with weak or absent theta-modulation reacted to stimulation by stimulus-locked single spike discharges which followed up to high frequencies in both conditions.     

Functional regulation of the neuronal activity of the rat hippocampus transplanted into the rabbit septum

Embryonal tissue of the rat hippocampus was grafted into dorsolateral septum of the rabbit after its disconnection from the hippocampus. Extracellular investigation of the grafted neurones was performed in chronic conditions 6 to 7 weeks later. The grafted neurones had some characteristics typical of the rat hippocampus (low spontaneous activity and presence of complex spikes, low-frequency rhythmic modulation--below 1 Hz, significant increase of activity level after physostigmine injection). Weak periodic theta-modulation, observed in spontaneous activity of some grafted neurones, became more stable and appeared in additional units after injection of physostigmine, under the influence of electrical stimulation of the septum and reticular formation and in response to sensory stimulation of the host animal. Its frequency was 4.5-6.0 Hz, as in the host septum. Microelectrode investigation for 5-10 days provoked lymphocyte infiltration of the grafts, which was not observed in the grafts not subjected to such treatment.     

Supernumerary chromosomes,Robertsonian rearrangement and variability of the sex chromosomes inNectomys squamipes (Cricetidae,Rodentia)

Chromosomes were analyzed in 91 specimens ofNectomys squamipes, collected in three distinct geographic regions of Brazil. Chromosomal polymorphism due to supernumerary chromosomes, Robertsonian rearrangement and variation of sex chromosomes is reported. Samples collected in the northeastern region had 2n=52, FN=52; 2n=53, FN=54; 2n=54, FN=56; 2n=55, FN=56; 2n=56, FN=56; 2n=57, FN=57. The specimens from the southeastern and southern regions showed 2n=55, FN=56; 2n=56, FN=56; 2n=57, FN=58; 2n=58, FN=60; 2n=59, FN=62. Seven different types of supernumeraries were observed. These vary in size, morphology and C-banding characteristics.The Brazilian populations ofNectomys squamipes are composed of at least two basic entities, one with 2n=52 and its increasing series up to 2n=55, the other with 2n=56, increasing to 2n=59. Both entities are characterized by variation from 0 to 3 supernumeraries. The X and Y chromosomes present polymorphisms in terms of the size, shape as well as the distribution of constitutive heterochromatin.This research was supported by CNPq-PIG (Projeto Integrado de Genética) and is part of the Rodent Cytogenetic Colaborative Study, developed by different Laboratories in Brazil.     

Chemical Composition,Anthelmintic Activity,and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC₅₀=0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.