Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.     

Responses of barley,pea, and maize to inoculation with different vesicular-arbuscular mycorrhizal fungi in irradiated soil

Summary The efficiency of different VAM fungi was investigated by inoculating barley, pea, and maize with different VAM fungi in irradiated soil in pots buried in the field. VAM frequency, growth and nutrient uptake were measured. In barleyGlomus epigaeus (CA) andG. macrocarpus (CA) were the most efficient out of 11 tested species and increased yield of grain by 24% and 21%, though they were not significant according to oneway analysis of variance. In pea, yield of grain was significantly increased from 46% to 104% (mean=68%) by 7 out of 10 tested species and by 105% by application of P fertilizer. The most efficient species wereG. epigaeus (CA),G. mosseae (GB), andG. etunicatus (CA). In maizeG. mosseae (GB) andG. caledonius (DK) increased total yield significantly by 59% and 47% in one experiment and in another experiment yield of cob was increased by 68% byG. mosseae (GB), 72% byG. caledonius (DK), and by 153% by application of P fertilizer. This experiment demonstrated that responsiveness to inoculation by VAM fungi differed among plant species, and that efficiency of different VAM fungi differed.     

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK_a values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO⁻CH₂NH₂⁺CH₂PO₃²⁻, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K_i = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K_i′ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate].     

The interstitial cells in the renal medulla of rat,rabbit, and gerbil in different states of diuresis

Summary The inner zone of the renal medulla of rats, gerbils, and rabbits was investigated to determine whether or not there are any characteristic ultrastructural differences between the interstitial cells of these species. The effects on the interstitial cells of water deprivation and water loading were also investigated.In all three species, the Type 1 interstitial cells, the lipid containing cells, were abundant and their distribution and topographical relations as well as their general ultrastructure were similar. The previously reported significantly higher frequency in desert rats could not be confirmed. Although the lipid droplets of the interstitial cells were smaller in gerbils and rabbits when compared to rats, their fine structure was similar. Their electron dense outer zone was sometimes associated with a granular material and/or a lamellar material with a periodicity of about 40 Å resembling phospholipid myelin figures.Water-loaded rats showed a considerable increase in the number of lipid droplets when compared to dehydrated or untreated animals. In contrast, the interstitial cells of waterloaded gerbils and rabbits were depleted of lipid droplets.We are indebted to Professor A.B. Maunsbach for valuable discussions and criticism and to Mrss. Hanne Weiling and Birthe Overgaard for competent technical assistance. The gerbils used in this study were a gift from Leo AB, Helsingborg, Sweden. This study was supported by the Danish Medical Research Council (J. nos. 512-1067, 512-1545, 512-3633)     

Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.     

Quantitative studies on brown algal phenols. I. Estimation of absolute polyphenol content of Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.)

Methods for estimating the absolute polyphenol content of the brown algae Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.) have been developed and tested. Polyphenols were extracted almost quantitatively from Ascophyllum nodosum using aqueous acetone, whereas this procedure was somewhat less efficient with Fucus vesiculosus. Colorimetric methods based on the Folin-Denis reagent, Brentamine Fast Red 2G Salt, and vanillin-H₂SO₄ were applied to acetone-free extracts for determination of polyphenol content relative to suitable reference compounds. Gravimetric methods based on hide powder and on haemoglobin were employed to derive ‘estimation factors’ (EFs) which allow calculation of the absolute polyphenol content from the relative polyphenol content. The values calculated for absolute polyphenol content are considered to be reasonably accurate, despite imprecisions in the methods and despite often large standard deviations, and re-emphasize the potential physiological and ecological significance of brown algal polyphenols. Although the precise EFs calculated here are not valid for other brown algae, the methods are considered to be generally applicable to other Phaeophyceae.     

Identification of the primary lesion in a protoporphyrin accumulating mutant of Chlamydomonas reinhardtii

Summary A group of chlorophyll deficient mutants (br _s mutants) of Chlamydomonas accumulates protoporphyrin and has poorly developed chloroplast membrane systems (Wang et al. 1974). In order to determine whether a poorly developed chloroplast membrane system is the reason for, or the result of, the inability of the br _s mutants to metabolize protoporphyrin to chlorophyll, a second mutation was selected which restored chlorophyll synthesis in br _s mutants. One such double mutant (br _s-2 g-4) was analyzed. The double mutant br _s-2 g-4 has partially restored chlorophyll synthesis, but has defective photosystem II and photosystem I electron transport as well as abnormal chloroplast ultrastructure. Since these defects are not present in cells carrying only the g-4 mutation, they are presumed to be caused by the br _s-2 mutation. It is concluded that a defect in chloroplast membrane development resulting from the br _s-2 mutation causes an apparent defect in magnesium chelation by protoprophyrin. This is consistant with evidence that chlorophyll biosynthesis from magnesium protoporphyrin to chlorophyll takes place on the chloroplast membranes.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.