Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

SPP1-mediated plasmid transduction.

The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described.     

Current-voltage curve of electrogenic Cl− pump predicts voltage-dependent Cl− efflux inAcetabularia

Summary The current-voltage relationship of carrier-mediated, passive and active ion transport systems with one charge-carrying pathway can exactly be described by a simple reaction kinetic model. This model consists of two carrier states (one inside, one outside) and two pairs (forwards and backwards) of rate constants: a voltage-dependent one, describing the transport of charge and a voltage-insensitive one, summarizing all the other (voltage-independent) reactions. For the electrogenic Cl^– pump inAcetabularia these four rate constants have been determined from electrical measurements of the current-voltage relationship of the pump (Gradmann, Hansen & Slayman, 1981;in: Electrogenic Ion Pumps, Academic Press, New York). The unidirectional Cl^– efflux through the pump can also be calculated by the availiable reaction kinetic parameters.³⁶Cl^– efflux experiments on singleAcetabularia cells with simultaneous electrical stimulation (action potentials) and recording, demonstrate the unidirectional Cl^– efflux to depend on the membrane potential. After subtraction of an efflux portion which bypasses the pump, agreement is found between the measured flux-voltage relationship and the theoretical one as obtained from the reaction kinetic model and its parameters from the electrical data.     

Prostaglandins do not modulate the positive chronotropic and inotropic effects of sympathetic nerve stimulation and injected norepinephrine in the isolated blood perfused canine atrium

In isolated canine atrium, perfused with blood from a donor dog, the infusions of both prostaglandins (PG)I₂ and E₂ (0.1–1 μg/min) into the sinus node arterial cannula neither altered the sinus rate and developed tension nor the positive chronotropic and inotropic responses elicited by either electrical stimulation or by injected norepinephrine. Infusion of arachidonic acid (10–100 μg/min), a precursor of PGs, or indomethacin (15–20 μg/min), an inhibitor of PG synthesis, into the sinus node arterial cannula also failed to alter the increase in sinus rate or developed tension produced by either adrenergic stimulus in the isolated atria. When arachidonic acid, 100–300 μg/kg or PGI₂, 1 μg/kg, were injected into the jugular vein of the donor dog, they produced a fall in systemic blood pressure; this effect of arachidonic acid but not of PGI₂ was abolished by indomethacin, 1 mg/kg. During administration of either arachidonic acid or indomethacin to the donor dog, the positive chronotripic and inotropic responses to adrenergic stimuli in the isolated atria also remained unaltered. These data indicate that PGs do not modulate adrenergic transmission in the blood perfused canine atrium.     

Electric field-induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenience

Using an electric field pulse technique, we induced fusion between vacuoles and protoplasts of Kalanchoë daigremontiana , between protoplasts from etiolated and green leaf mesophyll, and between mesophyll protoplasts from plants of different physiological properties ( Avena sativa : C₃ mechanism of photosynthesis, Kalanchoë daigremontiana : crassulacean acid metabolism). Close membrane contact amongst protoplasts or between protoplasts and vacuoles (as required for fusion) was achieved by the application of an alternating, non-uniform electric field to the suspension. Due to the dielectrophoresis effect the cells attach to each other along the field lines. The fusion process is initiated by the injection of an electric field pulse of high intensity and short duration (μs range). The field intensity has to be sufficiently high to induce reversible breakdown in the area of close membrane contact. After the application of the field pulse, the fusion process is initiated and completed within seconds to a few minutes, depending on the material investigated.
Fusion occurs between protoplasts and vacuoles as well as between protoplasts of different species. Both tonoplast and plasma membranes completely intermingled, indicating that in contrast to suggestions in the literature these membranes are compatible. Furthermore the cytoplasms of etiolated and green protoplasts obviously do not mix after fusion is completed, as etioplasts and chloroplasts kept separated from each other. In all experiments the volume of the fusion product equalled the sum of the compartments that underwent fusion. The wide spectrum of possible applications resulting from these fusion experiments in relation to metabolic problems is discussed.     

Destruction and reconstitution of the dictyosome in the chrysophycean flagellate,Poterioochromonas malhamensis,after heat-shock,and other heat-shock effects

Summary The viability of the chrysophycean flagellate,Poterioochromonas malhamensis, was examined after treatment of the cells with high temperatures. The fine structure of the cells was studied after heat-shock (42 °C, 16 minutes). Heat injury effects are visible at the nucleus, the chloroplast (distortion of the thylakoids), the mitochondria (increased occurrence of matrical crystalline inclusions) and, especially, at the dictyosome which is completely destroyed into some single vesicles and remnants of cisternae. Most damages are repaired within one hour; the reconstitution of the dictyosome takes 3–6 hours. It is inhibited severely by actinomycin D.The effect on the dictyosome reflects its labile position in the endomembrane system. The dependence of its reconstitution on protein synthesis indicates that membrane components are destroyed by the heat-shock.     

High frequency fusion of plant protoplasts by electric fields

Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 m). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This pearl chain arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 s), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.Presented at II Congress FESPP, Santiago de Compostela, Spain, 27.7.–1.8.1980, and Gordon Research Conference of Bioelectrochemistry, Tilton, New Hampshire, USA, 4.8.–8.8.1980     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

Infant development in the lion-tailed macaque,Macaca silenus (Linnaeus): The first eight weeks

Infant development in lion-tailed monkeys was observed during the first eight weeks after birth. A rapid decrease in the time spent in the closest association by the mother and the infant was seen between the first and second weeks. Between the second and fifth week there was a somewhat steady state with only marginal decrease in the time spent by the infant out of lap of the mother. However, during this time, a rapid decline in maternal cradling and restraining behaviour was seen. From the sixth week onwards, there was a rapid increase in the time spent by the infant out of contact with the mother. Independence of the infant during troop progression was seen only towards the eighth week, that too only marginal. Among the troop members the subadult females and the juvenile females showed the maximum interest in the infant. The former spent much more time than the juveniles interacting with the infant. The adult females showed only little interest in the infant while the adult male showed no interest at all. Infant snatching by subadult females and juvenile females was seen on many occasions. The infant remained in the possession of them from less than 1 min to more than 2 hr. This behaviour tended to decrease towards the sixth week.