Storage of embryonically transcribed poly(A) RNA and its utilization during metamorphosis of the hydroidHydractinia echinata

Summary The formation of tentacles and stolons during metamorphosis is severely disturbed if inhibitors of mRNA metabolism are applied during certain phases of development. The periods of sensitivity to -amanitin are late gastrulation and the disk stage of metamorphosis. A cordycepin sensitive phase exists during the first hour of metamorphosis. In all drug sensitive phases an enhanced poly(A) synthesis is found indicating increased mRNA metabolism in these stages. Pulse-chase experiments show that planula larvae store a poly(A)-rich RNA population sedimenting between 28–18s. These long living molecules are of embryonic origin, are located in RNP particles and are degraded during metamorphosis. The particles in question appear to be stored mainly in interstitial cells. In early metamorphosis no uridine is incorporated but labelled poly(A) is added to preexisting molecules.     

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.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Classification of amphipod compound eyes — The fine structure of the ommatidial units (Crustacea,Amphipoda)

Summary The ultrastructure of the compound eyes of 13 amphipod species has been investigated. An amphipod type of compound eye can be characterized by the constellation and consistency of a number of morphological features, most of which are also found in other compound eyes. The amphipod eye falls into four sub-categories (types). The ampeliscid type has a tripartite aberrant lens eye; the lysianassid type has a reduced or no dioptric apparatus and a hypertrophied rhabdom; the hyperid type possesses a large number of ommatidial units with long crystalline cones and dark instead of reflecting accessory pigment; and finally, the gammarid type can be interpreted as a generalized amphipod type. The lysianassid type is adapted to low light intensities and demonstrates convergent development with the compound eyes of other deep-sea crustaceans. The ampeliscid type is more similar to the gammarid type. The type characterization of the amphipod compound eye might well serve as a basis and incentive for functional studies also revealing adaptational mechanisms.This paper is dedicated to Professor Erik Dahl on his 65th birthday and retirement from the Chair of Structural Zoology, Department of Zoology, University of LundThe investigation has been supported by grants from the Swedish Natural Science Research Council (Grants 2760-009 and 009-43). Our thanks are due to the staffs of the marine biological stations in Espegrend (Norway) and Kristineberg (Sweden) and of the research vessel Jean Charcot, Brest, France. The skilled technical assistance of Mrs. Rita Wallén and Miss Maria Walles is gratefully acknowledged     

Computer control of fermentation processes

A review of computer control of fermentation processes is presented. Hardware and software technologies that have been used to implement computer control are discussed. This includes instrumentation, interfacing techniques, computer hardware configurations, data logging and documentation, displays and man-machine interaction, low-level control, back-up and error detection and programming techniques. Advanced control of fermentation processes with the utilization of modern control techniques is also presented. This topic is divided into steady state optimization and dynamic optimization. Finally, on-line estimation of bioreactor parameters for feedback control is presented.     

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10⁴ [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L -leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L -leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L -leucine. The kinetic constants V_max and K_m of enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L -leucine was produced continuously out of α-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

Modulation of the G0 to S phase transit time by insulin: Potential involvement of protein phosphorylation

BHK fibroblasts can be growth arrested by incubation in low serum (0.1%) medium. Growth is initiated by incubating cells in high serum (10%) medium. We have found that if the quiscent cells in low serum medium are incubated with insulin, the G₀ to S transit time is decreased by two to six hours when serum (10%) is added back to the culture. The effect of insulin treatment of quiescent cells on the cellular phosphoprotein profile was examined. It was found that insulin stimulated the phosphorylation of a 96,000 dalton cytosol protein. This protein is also intensely phosphorylated in proliferating cells and may be one of the critical intracellular events to occur when a cell initiates growth.     

Perifused monolayer cultures of rat hepatocytes as an improved in vitro system for studies on ureogenesis

A perifusion system has been developed for cultivation of adult rat hepatocytes, permitting continuous supply of medium to the cell monolayer instead of periodical changes as used in conventional culture technique (discontinuous culture). Additionally, a modification of Waymouth's MB 752/1 medium is described, which favoured the expression of several metabolic and regulatory events mentioned below and supported the maintenance of several enzymes involved in nitrogen metabolism. The improved nutritional conditions accelerated early monolayer formation and metabolic recovery, and favoured long-term cultivation of metabolically active and hormone responsive hepatocytes. Urea formation from substrates contained in the medium was found to be 2- to 3-fold higher and preserved for a considerably longer time than with discontinuously cultured cells, and was further enhanced by addition of tryptose phosphate broth or 4 mM NH₄Cl even after 10 days in culture. In the presence of glucagon (10⁻⁷ M) the urea production was more than doubled during a 24 h incubation period on the 4th day. Pretreatment with this hormone for 24 h also markedly stimulated the capacity of perifused cells for ureogenesis. Concomitantly, a rise in arginase activity up to 2-fold could be measured in response to glucagon, which was largely suppressed by simultaneous presence of leucine in concentrations between 5 and 10 mM.