Stimulative effect of somatostatin on cell proliferation in cultured chondrocytes

We have earlier demonstrated that human growth hormone stimulates DNA synthesis and proteoglycan production in cultured chondrocytes. The present study is concerned with the effects of somatostatin and other neuropeptides on cell proliferation by cultured rat rib growth plate chondrocytes. Chondrocytes were isolated from the growth plates by collagenase digestion and cultured as monolayers in multiwell plates. The cells were allowed to attach overnight and subsequently incubated for 24 h under serum-free conditions to establish growth arrest. Somatostatin and other peptides were then added and the cultures were incubated for 18 h. Finally, the cultures were labelled for 6 h with tritiated thymidine in the presence of peptide. For screening purposes, the effect on DNA-synthesis was assayed as incorporation of [3H]-thymidine into acid-insoluble material. For a more exact estimate, parallel cultures were prepared for autoradiography and the fraction of labelled nuclei was determined by counting. Among the peptides we tested (somatostatin, GRF, TRH, SP, mENK, PHI, VIP, hCT) only somatostatin had any discernible effect on DNA synthesis, with an apparently optimal effect at 10 fM. This concentration is well within the range found in various tissues in vivo and suggests a physiological role for somatostatin in chondrocyte growth regulation. Further experiments are required, however, to clarify by which mechanism somatostatin influences the cells and whether the peptide interacts with other growth factors such as the IGFs.     

Evidence for dose-dependent effects on plant growth by Stenotrophomonas strains from different origins

AIMS: To assess the influence of Stenotrophomonas on plants, the interaction of 16 Stenotrophomonas strains from clinical and environmental sources with strawberry plant seedlings was analysed. METHODS AND RESULTS: In vitro, all Stenotrophomonas strains influenced plant growth when applied to seedlings. Whereas most of the Stenotrophomonas strains promoted root growth and hair development, a statistically significantly negative influence on the length of stem was found. Although strains from a clinical origin also showed statistically significant effects on plants, this was generally lower when compared with environmental strains. For three selected strains, a strong dose-dependent effect was observed for all parameters. In vitro, a correlation was found between plant growth promotion and production of a plant growth hormone, indole-3-acetic acid (IAA). Xanthomonas campestris, a phylogenetically very closely related species to Stenotrophomonas, was used as a phytopathogenic control. It too confirmed the reduction of plant growth in this in vitro system. CONCLUSIONS: Independent of their origin, Stenotrophomonas strains can produce IAA in vitro and subsequently, influence plant growth. The effect of Stenotrophomonas presence on plants was dose-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-dependent effect of Stenotrophomonas, a bacterium of both biotechnological and medical interest, is of great interest for biocontrol applications of plant-associated strains. This paper is the first report that clearly demonstrates the phytopathogenic capacity of Stenotrophomonas.     

Solubilization of starch synthetase bound to Oryza sativa starch granules

Starch synthetase was solubilized from purified starch granules of ripening grains of rice at the midmilky stage. The procedure consisted of making the granules amorphous and dispersing the amorphous starch by sonication in 75% dimethysulfoxide. A starch synthetase-amylose complex was isolated by discontinuous sucrose density gradient centrifugation, which does not require added primer and can utilize both ADP glucose and UDP glucose. A starch-free protein fraction was obtained by treatment with sodium dodecyl sulfate and β-mercaptoethanol.     

Evolutionary origins of mechanosensitive ion channels

According to the recent revision, the universal phylogenetic tree is composed of three domains: Eukarya (eukaryotes), Bacteria (eubacteria) and Archaea (archaebacteria). Mechanosensitive (MS) ion channels have been documented in cells belonging to all three domains suggesting their very early appearance during evolution of life on Earth. The channels show great diversity in conductance, selectivity and voltage dependence, while sharing the property of being gated by mechanical stimuli exerted on cell membranes. In prokaryotes, MS channels were first documented in Bacteria followed by their discovery in Archaea. The finding of MS channels in archaeal cells helped to recognize and establish the evolutionary relationship between bacterial and archaeal MS channels and to show that this relationship extends to eukaryotic Fungi (Schizosaccharomyces pombe) and Plants (Arabidopsis thaliana). Similar to their bacterial and archaeal homologues, MS channels in eukaryotic cell-walled Fungi and Plants may serve in protecting the cellular plasma membrane from excessive dilation and rupture that may occur during osmotic stress. This review summarizes briefly some of the recent developments in the MS channel research field that may ultimately lead to elucidation of the biophysical and evolutionary principles underlying the mechanosensory transduction in living cells.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.     

Effects of univalent cations on the activity of particulate starch synthetase

An investigation was made to determine the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCl. Rubidium, cesium, and ammonium were 80% as effective as potassium while sodium and lithium were respectively 21% and 8% as effective as potassium. The K_A for potassium was determined to be 6 mM. In the case of the particulate starch synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, considerable stimulation of enzyme activity was obtained by the addition of potassium to the reaction mixture. In these studies, low enzyme activity was observed in the absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been negative.     

The effect of potassium on germinating wheat embryos at different viability levels

The effect of potassium administration in early germination of wheat (Triticum durum L., cv. Appulo) was studied by incubating isolated embryos at different viability levels with 1 mM KC1. The stimulation of germination and growth in aged embryos by K⁺ was accompanied by a significant increase in (³H) leucine incorporation into protein and in phospholipid synthesis as evidenced by TLC analysis of embryo phospholipids extracted after 24 h of imbibition. The metabolic improvement mediated by the cation would in turn repair aging damage occurring at subcellular sites including membranes.     

Chiroptic recognition of potassium ion

The high specificity in the recognition and specific binding of potassium ion by the depsipeptide valinomycin (VM) is exploited for its recognition and quantitation using both circular dichroism (CD) and optical rotation dispersion (ORD). The specific rotation of VM is comparably small (2.34 deg ml g(-1) cm(-1)), so that an 8 microM (= 8.89 mg ml(-1)) solution of VM in 95% ethanol rotates polarized light of Lambda = 426 nm passing a 2 cm cuvette by 0.076 degrees only. It is shown, however, that VM undergoes large changes in both ORD and CD on binding to potassium ion. VM, potassium ion and the anionic dye merocyanine 540 form a ternary complex (VM/K/MC) which displays an induced CD with a positive maximum at 488 nm and a negative maximum at 470 nm. The ternary complex also displays fluorescence that is weaker by about 30% when compared to that of the dye alone. The induced CD of the ternary complex is interpreted in terms of the large conformational change which VM is known to undergo on binding potassium ion, thereby forming the prerequisite for a van der Waals interaction between its outwardly directed lipophilic domains and the lipophilic domains of the anionic dye. The method is likely to be applicable to the fluorescent detection of all kinds of ions for which chiral receptors are known, e.g. in studies on the role of ions in biological systems including ion channels.     

Isoenzymes of soluble starch synthetase from Oryza sativa grains

Two isoenzymic fractions of soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase were obtained from developing (non-waxy) rice grains by gradient elution through DEAE-cellulose. After Sephadex G-200 chromatography, fractions I and II were electrophoretically homogeneous and have MW values of 110000 and 69000, respectively. Sodium dodecyl sulfate gel electrophoresis of fraction I produced five bands with MW of 12000, 26000, 50000, 70000, and 105000 while fraction II gave two bands with MW of 12000 and 22000. Fraction II, which contains 1·7% carbohydrate, was active in the absence of added primer while fraction I, which does not contain carbohydrate, required primer.     

Quantification of starch in plant tissues

This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide range of starch contents. Measurements on multiple samples can be completed within a day.     

Enzymic mechanism of starch synthesis in ripening rice grains VI. Isozymes of starch synthetase

The DEAE-cellulose column chromatography has shown two differentforms of starch synthetase, which are referred to as fractionsI and II in extracts of rice seeds (non-waxy and waxy varieties)harvested at the milky stage. Similarly treated leaf extractsof rice (non-waxy) and maize (non-waxy) also demonstrate dieexistence of two major isozyme fractions. In all enzyme preparationstested, ADP-glucose was the sole glucosyl donor and UDP-glucosewas totally inactive. Rechromatography, on a DEAE-cellulosecolumn, of two enzyme fractions (I and II) separated from non-waxyrice seed extracts did not alter their elution patterns. Someof their enzymic properties were compared, in particular, theirglucosyl-acceptor (primer) specificities. Regardless of potentamylase activities in the two fractions, notable differenceswere observed in that fraction I utilized the long-chain oligosaccharides[maltododecaose] and various types of high molecular -glucansmore readily than fraction II. However, short-chain oligosaccharides[maltose, maltotriose and maltotetraose] were utilized morereadily by fraction II than by fraction I. A possible role forthe two starch synthetase isozymes in starch synthesis in plantcells is discussed. (Received January 5, 1971; )     

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.     

Energetic efficiency of different mechanistic models for potassium ion uptake in lower eukaryotic cells

Different mechanistic models for potassium ion uptake are analyzed by an equilibrium-thermodynamic formalism in terms of their comparative efficiency in setting chemical potential differences of the potassium ion of different magnitudes across the plasma membrane of lower eukaryotic cells. The possible adaptive advantages for a multimode mechanism(s) operating in alternative modes depending on the physiological and/or environmental conditions of the cells are discussed.     

Atomic force microscopy of pea starch: origins of image contrast

Atomic force microscopy (AFM) has been used to image the internal structure of pea starch granules. Starch granules were encased in a nonpenetrating matrix of rapid-set Araldite. Images were obtained of the internal structure of starch exposed by cutting the face of the block and of starch in sections collected on water. These images have been obtained without staining, or either chemical or enzymatic treatment of the granule. It has been demonstrated that contrast in the AFM images is due to localized absorption of water within specific regions of the exposed fragments of the starch granules. These regions swell, becoming "softer" and higher than surrounding regions. The images obtained confirm the "blocklet model" of starch granule architecture. By using topographic, error signal and force modulation imaging modes on samples of the wild-type pea starch and the high amylose r near-isogenic mutant, it has been possible to demonstrate differing structures within granules of different origin. These architectural changes provide a basis for explaining the changed appearance and functionality of the r mutant. The growth-ring structure of the granule is suggested to arise from localized "defects" in blocklet distribution within the granule. It is proposed that these defects are partially crystalline regions devoid of amylose.