Circadian rhythm mutants of the prokaryoticSynechococcus RF-1

Synechococcus RF-1 established circadian rhythms in nitrogen fixation and leucine uptake when growing in a diurnal light/dark regimen. The rhythms persisted in subsequent uniform light/light conditions. In order to analyze the circadian rhythm at the genetic level, mutants were induced by N-methyl-N-nitro-N-nitrosoguanidine and then isolated by procedures with the circadian nitrogen-fixing rhythm as a selecton marker. Characterization of the mutants with respect to the circadian rhythm indicated that some mutants were abnormal only in the nitrogen-fixing rhythm, while some simultaneously lost the ability to establish the nitrogen-fixing and leucine-uptake rhythms. The physiological properties of the circadian rhythm were compared. The genetic potential of the mutants that were abnormal in both rhythms is emphasized.     

“Differentiation Induction” culture of human leukemic myeloid cells stimulates high production of macrophage differentiation inducing factor

A suitable procedure for the production of human monokines was defined as differentiation-induction culture. Human monocytic leukemia THP-1 cells were well-differentiated from nonfunctional promonocytes into macrophage-like cells by the induction with a combination of mezerein, retinoic acid, and aMycoplasma fermentans extract. The differentiated THP-1 cells secreted a high amount of macrophage differentiation-inducing factor (DIF) activity and concomitantly produced other known monokines, such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and interleukin-1 (IL-1), into the medium. These results suggest that other novel human monokines may also be found in the conditioned medium of THP-1 cells induced by the differentiation-induction culture conditions defined in this study. Macrophage DIF was purified to homogeneity and NH₂-terminal amino acid sequence analysis revealed that macrophage DIF is very similar or identical to human leukemia inhibitory factor (LIF). The cDNA encoding human LIF was isolated using the polymerase chain reaction, and a clone producing 3.7 g/10⁶ cells day recombinant LIF was selected from Chinese hamster ovary (CHO) cells which were transfected with the LIF cDNA. The recombinant LIF production in CHO cells was quantified using MTT reduction assay with M1 cells.     

The aerobic nitrogen-fixingSynechococcus RF-1 containing uncommon polyglucan granules and multiple forms of α-amylase

The aerobic nitrogen-fixing unicellular cyanobacteriumSynechococcus RF-1 (pcc 8801) contains numerous irregular polyglucan granules ranging in size from 0.1 to 0.4 m. These morphologically unusual polyglucan granules are not found in the sheathed nitrogen-fixing unicellular cyanobacteriumGloeothece RF-6 (pcc 8803). Two forms of -amylase, ethyleneglycolbis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-sensitive and EGTA-insensitive, were found in theSynechococcus RF-1. In addition to their EGTA sensitivity, the two forms of -amylase also differed in their reactive pH range and in their zymogram disc gel electrophoresis. Each form of -amylase was stable and constant in concentration through a diurnal light/dark cycle.     

Isolation of two thioredoxins from the cyanobacterium Synechococcus 6301

Two different thioredoxins designated as thioredoxin A and B have been isolated from the cyanobacterium Synechococcus 6301. Methods for large scale purification of these thioredoxins were developed. Thioredoxin B has been purified to homogeneity; it has a molecular weight of 11,800 and an isoelectric point of 4.6. The following K _m data were obtained for this thioredoxin; a) in the PAPS-sulfotransferase assay of Synechococcus 6301: 10.7 M; b) in the fructose-1-6-bisphosphatase assay of Synechococcus 6301: 1.7 M; c) in the APS-sulfotransferase assay of Chroococcidiopsis 7203: 5.4M. Thioredoxin A has an isoelectric point of 4.1 and it is active in the PAPS-sulfotransferase and fructose-1-6-bisphosphatase of Synechococcus 6301; it is not active in the APS-sulfotransferase of Chroococcidiopsis 7203.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Somatic embryogenesis and plant regeneration from protoplasts of Cichorium intybus L. x Cichorium endivia L.

Somatic embryos and adult plants were regenerated from mesophyll protoplasts of a clone of chicory 474 (Cichorium intybus L. x Cichorium endivia L.). Embryos were obtained in three different ways:

Circadian complexes: Circadian rhythms under common gene control

Summary Circadian rhythms for food and water consumption were measured in five inbred strains of mice under a photoperiod of 16 h light and 8 h dark (16:8 LD), and under constant light (LL).Significant strain differences were observed which indicate that a common gene difference, or set of differences inMus musculus influences both the phase angle () associating the rhythms with the light-dark cycle, and the periods (_LL) of circadian rhythms for food and water consumption. The biological clock mechanism influenced by this genetic variance is common to both food and water circadian rhythms, and differs among the five inbred strains. A positive genetic correlation was observed between the phase angle () and the period (_LL) of each rhythm. This observation can be understood in terms of a functional relationship between phase and period proposed by Pittendrigh and Daan (1976b) for the entrainment of a circadian oscillator by a light-dark cycle in nocturnal rodents.These results suggest that circadian rhythms for food and water consumption in mice are regulated by a common physiological mechanism, and would respond to natural selection as a single circadian complex under common gene control.     

Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll

Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-d- and 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[-d-glucuronosyl-(12)-d-glucuronide] (R₂) and luteolin 7-O-[-d-glucuronosy 1 (12) -d-glucuronide]-4-O--d-glucuronide (R₁), as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific -glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific -glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the triglucuronide is deglucuronidated, this being the first step for further turnover.Dedicated to Professor Ludwig Bergmann, Botanisches Institut der Universität zu Köln, on the occasion of his 65th birthday     

Pinealectomy abolishes the circadian rhythm of migratory restlessness

Summary Pinealectomy of White-throated Sparrows (Zonotrichia albicollis) free-running under constant conditions in dim light abolishes the circadian rhythm of nocturnal spring and fall migratory restlessness (Zugunruhe) as well as the rhythm of summer daytime locomotor activity (Pigs. 1 and 2). Rhythmicity persists in sham-operated birds. Pinealectomized birds are synchronized by a light cycle but their activity rhythm decays to arrhythmicity when they are released from entrainment into constant dim light. The pineal of the white-throat seems essential for the expression of circadian rhythms of both daytime activity and migratory restlessness. These findings support the hypothesis that the avian pineal is fundamentally involved in circadian organization.     

Compartmentation and flux characteristics of ammonium in spruce

Using ¹³NH ₄ ⁺ as a tracer, compartmental analyses for NH ₄ ⁺ were performed in non-mycorrhizal roots of intact Picea glauca (Moench) Voss. seedlings at four different concentration regimes of external NH ₄ ⁺ ([NH ₄ ⁺ ]_o), i.e. 0, 10, 100, and 1500 M. Three kinetically distinct compartments were identified, with half-lives of exchange of approximately 2 s, 30 s, and 14 min, assumed to represent surface adsorption, Donnan free space, and cytoplasm, respectively. No significant differences were found in half-lives of exchange with changes in [NH ₄ ⁺ ]_o. Influx was calculated to be 0.96 mol·g^–1·h^–1 in N-deprived plants (measured at 10 M [NH ₄ ⁺ ]_o), while under steady-state conditions it was 0.21 mol·g^–1h^–1 at 10 M [NH ₄ ⁺ ]_o, 1.96 mol·g^–1h·^–1 at 100 M [NH ₄ ⁺ ]_o, and 6.45 mol·g^–1·h^–1 at 1.5 mM [NH ₄ ⁺ ]_o. Efflux measured over the same range constituted approximately 9% of influx in N-deprived plants, 10% at 10 M, 28% at 100 M, and 35% at 1.5 mM [NH ₄ ⁺ ]_o. Cytoplasmic [NH ₄ ⁺ ] was estimated at 6 m M in N-deprived plants, 2 mM at 10 M [NH ₄ ⁺ ]_o, 14 mM at 100 M, and 33 mM at 1.5 mM. Free-space [NH ₄ ⁺ ] was 84 M, 50 M, 700 M, and 8 mM, respectively. In comparison with previously published data on fluxes and compartmentation of NO ₃ ^– in white-spruce seedlings, results of this study identify a pronounced physiological preference of this species for NH ₄ ⁺ over NO ₃ ^– as an inorganic N source in terms of uptake and intracellular accumulation. The significant ecological importance of this N-source preference is discussed.The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. P. Poon for essential assistance in experiments.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid