Isolation,culture and callus regeneration of protoplasts of wild and cultivated Helianthus species

Protoplasts were isolated from seedling roots, hypocotyls, and cotyledons of four cultivars of Helianthus annuus and from leaves of axenic shoot cultures of the wild species H. praecox, H. scaberimus and H. rigidus. Optimal culture conditions were established for the respective protoplast systems, using the agarose bead method of culture. Protoplast division was induced for all the species examined. In the case of the cultivars of H. annuus, hypocotyl and cotyledon protoplast division was sustained leading to callus formation, which in turn, could be induced to produce roots and organised meristematic regions in the presence of NAA and 6-BAP.Abbreviations 6-BAP 6-benzylaminopurine - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

An electron-spin-resonance spin-label study of the interaction of purified Mojave toxin with synaptosomal membranes from rat brain

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.     

Lead Poisoning in Gurkha Soldiers in Hong Kong

Investigation of an outbreak of lead-poisoning in 121 Gurkha soldiers showed that this was due to the contamination of chilli powder (cayenne pepper), a constituent of curry powder, with lead chromate. Comprehensive systems of food sampling are needed in developing communities.     

Microbial interactions: Population and sporulation studies ofBacillus sphaericus grown in association withErwinia atroseptica

A study was made of a phenomenon, previously reported, in whichBacillus sphaericus failed to sporulate in the usual peptone media, but would sporulate in these media when grown in association withErwinia atroseptica.Pure culture studies withB. sphaericus indicated that the stimulus driving the cells toward further vegetative growth and the resulting failure of the cells to sporulate was associated with the peptide fraction of peptone media; inhibition of sporulation could be reversed by reduction of the peptone level of the medium or by replacement of the peptone with known amino acids, with known amino acids and short-chain peptides, or with complete hydrolysates of casein.Population and sporulation studies were performed onB. sphaericus cultured inE. atroseptica spent medium and on mixed cultures of the two organisms. A variety of population and sporulation responses were obtained through alteration of the chemical and physical nature of the media byE. atroseptica, cultured alone or in mixed culture withB. sphaericus.It is suggested that removal of pro-vegetative peptides byE. atroseptica is responsible for the enhancement of sporulation observed inB. sphaericus in peptone media.From a thesis submitted to the Graduate School of the University of Maryland, by the senior author, in partial fulfillment of the requirements for the Ph. D. degree.     

Diversifications in the Tube Dilution Test for Antibiotic Sensitivity of Microorganisms

The tube dilution method of performing antibiotic sensitivity tests is commonly employed as an accurate method for defining the minimal inhibitory concentration in relation to pathogenic organisms. It is also used as a reference for comparing minimal inhibitory concentrations with the size of the zone of inhibition in the agar diffusion test. Although surveys have shown that there is no standardized method and technique of performing the tube dilution test, it is generally assumed that all of the diversified methods will yield the same results and interpretations. With the assistance of five experts, seven different tube dilution methods were compared; 16 antibiotics, and three organisms for each antibiotic, were used. The conclusions drawn are that, although the accuracy of a single method within its own confines is acknowledged, the minimal inhibitory concentrations and interpretations cannot be interpolated from one laboratory to another where a different technique is employed. The results are frequently discrepant. It is suggested that a uniform method be developed and promulgated for general use.