On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentrations of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP was not the reason of the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Phototropic fluence-response relations for Avena coleoptiles on a clinostat

Phototropism of Avena sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14° is expressed within 180 min following the single pulse; a final curvature of about 62° in about 240 min following five pulses without pre-irradiation; and a curvature of over 125° in 360 min following five pulses after the RL pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 pmol·cm^-2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol·cm^-2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5-min RL co-irradiation increases with fluence to a maximum of about 60° at about 10 pmol·cm^-2, and then decreases to 0° at about 200 pmol·cm^-2. Curvature induced by five BL pulses following a 2-h RL pre-irradiation increased with fluence from a threshold of about 0.05 pmol·cm^-2 to a maximum of 90° at about 10 pmol·cm^-2, and then gradually decreased with fluence to 50° at 1 000 pmol·cm^-2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation downstream of the photoreceptor pigment for phototropism, and that the additivive effect of pulsed BL is distinct from the potentiating effect of RL on phototropism. Thus, any degree of curvature from 0° to over 90° may be induced by a fluence in the ascending arm of what is traditionally called the first positive phototropic response.Abbreviations BL blue light - RL red light     

Cyclic AMP-stimulated protein kinase activity in rabbit peripheral myelin

Cyclic AMP-sensitive protein kinase activity has been found in suspensions of purified rabbit peripheral myelin. The enzyme phosphorylated the P₀, Y, X, P₁, and P₂ myelin proteins. Kinase activity, which was maximal at physiological pH, 2.5 mM Mg²⁺, and 2 M cAMP, was stimulated three-fold over basal levels by cyclic AMP. Addition of calcium or EGTA had no effect on the enzyme activity in the presence or absence of cyclic AMP. Cyclic GMP also did not stimulated endogenous or exogenous protein phosphorylation. Theophylline, an inhibitor of 3,5-cyclic nucleotide phosphodiesterase activity, increased protein kinase activity in the presence of cyclic AMP. These data show that PNS myelin proteins can be phosphorylated in situ by a protein kinase system whose activity is stimulated selectively by cyclic AMP.     

The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus<Emphasis Type="Italic"> Madracis</Emphasis>

Three species of the reef coral genus Madracis display skeletal isotopic characteristics that relate to depth, colony topography, and consequently to coral physiology. The joint interpretation of skeletal ¹³C and ¹⁸O provides information on the ecological plasticity and adaptation to depth of a coral species. Isotopic results are most easily understood in terms of kinetic effects, which reduce both ¹⁸O and ¹³C below isotopic equilibrium values, and metabolic effects, which only influence the skeletal ¹³C. Madracis mirabilis is adapted to depths shallower than 20 m, and shows the greatest range in kinetic effects and the strongest metabolic ¹³C enrichments caused by symbiont photosynthesis. Madracis formosa lives deeper than 40 m, and shows a reduced range of kinetic effects and relatively weak metabolic ¹³C enrichments. Madracis pharensis inhabits depths from 5 to >60 m, and does not attain the strength of kinetic effects of either of the other two species, apparently because it is not quite as well adapted to rapid growth at either extreme.     

In vitro propagation of the Greek olive cultivar `Chondrolia Chalkidikis'

The effect of basal media (Woody Plant Medium (WPM), Quoirin and Lepoivre (QL) and Olive Medium (OM)) and of various concentrations of cytokinins (6-benzyladenine (BA), zeatin, 6-(,-dimethylallyl-amino) purine (2iP)), solely or in combinations with each other and with gibberellic acid (GA₃), on in vitro shoot proliferation of the greek olive cultivar `Chondrolia Chalkidikis' was investigated. WPM proved to be the most effective one, resulting in better morphological appearance of the microshoots produced. The highest number of new microshoots/explant (1.68), with a 3.0 cm shoot height and a 4.2 proliferation rate, was obtained when this medium was supplemented with 20 M zeatin. The number of microshoots/explant and proliferation rate increased to 1.85 and 6.8, respectively, by using the combination of 5–20 M zeatin with 1 M BA, but shoot height was reduced. 2iP was the least effective of the cytokinins tested. Combination of 20 M zeatin with 10 M GA₃ affected positively shoot proliferation resulting in 1.80 microshoots/explant, 3.0 cm shoot height and 7.0 proliferation rate. However, the same concentration of GA₃ in combination with 1 M BA reduced the number of new microshoots/explant (0.48) as well as the proliferation rate (2.4), although shoot height remained nearly the same (2.7). The effect of indole-3-butyric acid (IBA), -naphthaleneacetic acid (NAA) and putrescine on root induction was also studied. A rooting percentage of up to 70% and 2.3 roots/microshoot were achieved by the combination of 12 M IBA + 3 M NAA, however, abscission of shoot tips and leaves appeared. Both variables were increased, up to 93% and 4.0, respectively, by the addition of 30 M putrescine in the medium, without any undesired side effect. After acclimatization, survival of rooted microshoots was high (90%) while that of non-rooted ones was low (30%).     

The structure of two distinct pancreatic amylase genes in mouse strain YBR

The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A and B, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage , followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II; another of 41 kb with a complete but different gene, PAN-I, plus a truncated gene, PAN-1; and finally, one of 23 kb with another truncated gene, PAN-2. Parts of the amino acid sequence of A and B have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II which establishes that this gene codes for B.This work was supported by the Danish Natural Science Research Council.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Process parameters and reusability of the free cell mass of Torulaspora delbrueckii for the production of L-phenylacetylcarbinol (L-PAC)

The effect of process parameters on the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using a yeast isolate identified as Torulaspora delbrueckii was studied. The maximum yield of L-PAC obtained was (331 mg) per 100 ml biotransformation medium (glucose 3%, peptone 0.6% and at pH 4.5) from 600 mg of benzaldehyde with 8 h of reaction at 30 ± 2 °C. Growing the organism in presence of 3% glucose reduced the biotransformation time to 120 min. Addition of 0.6% acetaldehyde (30–35%) lead to an increase in L-PAC yield to 450 mg%. Semi-continuous feeding of benzaldehyde (200 mg) and acetaldehyde (200 l) four times at 30 min intervals could produce 683 mg of L-PAC/100 ml biotransformation medium. Chiral HPLC analysis of purified L-PAC and PAC-diol showed 99% enantiomeric purity. The cell mass was found to be reusable for biotransformation up to nine times when benzaldehyde and acetaldehyde levels were maintained at (350 mg and 350 l)–(400 mg and 400 l). At concentrations from 450 mg and 450 l to 600 mg and 600 l, however the cell mass could give efficient biotransformation only during one use.     

Isolation and Properties of Cellobiase from Penicillium verruculosum

Cellobiase (-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i. e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose--lactone competitively inhibited cellobiase (K _i0.19 mM and 17 M, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.     

Cyclic GMP-activated channels of the chick pineal gland: Effects of divalent cations,pH, and cyclic AMP

Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 M Ca²⁺ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca²⁺ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg²⁺ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 M, 500 M, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 M cyclic AMP decreased the probability of channel activation caused by 20–100 M cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca²⁺, pH, or cyclic AMP.     

Dissection of a malting quality QTL region on chromosome 1 (7H) of barley

Malting and brewing are major uses of barley (Hordeum vulgare L.) worldwide, utilizing 30–40% of the crop each year. A set of complex traits determines the quality of malted barley and its subsequent use for beer. Molecular genetics technology has increased our understanding of genetic control of the many malting and brewing quality traits, most of which are quantitatively inherited. The objective of this study was to further dissect and evaluate a known major malting quality quantitative trait locus (QTL) region of about 28 cM on chromosome 1 (7H). Molecular marker-assisted backcrossing was used to develop 39 isolines originating from a Steptoe / Morex cross. Morex, a 6–row malting type, was the donor parent and Steptoe, a 6–row feed type, was the recurrent parent. The isolines and parents were grown in four environments, and the grain was micro-malted and analyzed for malting quality traits. The effect of each Morex chromosome segment in the QTL target region was determined by composite interval mapping (CIM) and confirmed and refined by multiple interval mapping (MIM). One QTL was resolved for malt extract content, and two QTLs each were resolved for -amylase activity, diastatic power, and malt -glucan content. One additional putative malt extract QTL was detected at the plus border of the target region by CIM, but not confirmed by MIM. All QTLs were resolved to intervals of 2.0 to 6.4 cM by CIM, and to intervals of 2.0 cM or less by MIM. These results should facilitate marker-assisted selection in breeding improved malting barley cultivars.     

Cyclic AMP stimulation of calcium efflux from kidney,liver, and heart mitochondria

Summary The effect of cyclic AMP on subcellular calcium turnover was studied in isolated kidney, liver and heart mitochondria. The calcium concentration of the incubating medium was determined by fluorometric methods after its separation by millipore filtration. Liver and kidney mitochondria take up calcium in exchange for H⁺ and lower the medium calcium to 1 to 40×10^–6 m in less than 2 min. Cyclic AMP produces an instantaneous release of calcium from mitochondria and a rise in the steady-state calcium concentration of the medium. A new medium calcium level of 0.7 to 3×10^–4 m is achieved in less than 3 sec and is proportional to cyclic AMP concentrations between 10^–7 and 3×10^–6 m. Cyclic AMP is inactive above 5×10^–6 m and below 10^–7 m. Cyclic IMP, 5 AMP, dibutyryl cAMP are inactive at any concentration. Cyclic GMP is active at 10^–5 m and competitively inhibits cyclic AMP action. The same staedy-state calcium level is reached from higher or, lower calcium concentrations, i.e. whether cyclic AMP is added before or after the addition of calcium to the mitochondrial suspension. At low calcium or phosphate concentrations, the calcium released by cyclic AMP is immediately reaccumulated by the mitochondria is less than 2 min with a further release of H⁺. This pulse can be repeated by sequential additions of cyclic AMP. The transient or sustained response to cyclic AMP depends on the medium calcium x phosphate product and presumably on the presence or absence of calcium phosphate precipitate inside the mitochondria. These results support the hypothesis that cyclic AMP regulates cytoplasmic calcium by controlling the mitochondrial calcium efflux rate. This mechanism may be involved in the regulation of calcium transport and in some hormonal effects mediated by cyclic AMP.     

The establishment of frequency dependent limits for electric and magnetic fields and evaluation of indirect effects

Summary The biophysical model described in this paper has been used as basis for the preparation of the German standards which determine and define limits of exposure to electric or magnetic fields below several MHz, including 50/60 Hz. The electric field strength within the tissue is considered decisive for the biological effect in the low frequency range. Threshold values of field strengths and current densities including biological effects are compared. It is possible to establish safe, dangerous and hazardous current density curves as a function of frequency. To define exposure limits, the field strength or current density causing injury should be reduced by a factor exceeding 100 in order to avoid well established biological effects. The electric and magnetic field strengths in the human environment are correlated with the corresponding electric current density induced in the human body. This enables safe, dangerous and hazardous levels of current density in the human body to be correlated with the external electric or magnetic field strength. Additionally to the direct field effects indirect effects must be considered. In the second part of this paper data on touch voltages and currents are summarized and evaluated with regard to their health risk. Furthermore, as an example for indirect effects the interference of electric and magnetic fields with pacemakers is considered.Review article by invitation of the editor     

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states

Summary Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2–3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200–800 ng dry weight. At saturating cyclic AMP concentrations (46 M) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 M) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 M) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.     

“Garden hunting” in the American tropics

Using faunal analysis, this article outlines a coastal mammalian harvesting pattern involving a few terrestrial mammals whose biomass appears to have been greater when associated with man than under natural conditions. Archeological evidence suggests that these animals fed regularly on cultivated crops and were hunted in house gardens and cultivated fields. By concentrating the supply of both carbohydrates and animal protein, garden hunting may have eliminated seasonality and scheduling problems. And because it artificially increased the biomass of selected animals, it may have functioned as a substitute for animal domestication.     

Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.