Comparison of 4-hydroxynzoate decarboxylase and phenol carboxylase activities in cell-free extracts of a defined, 4-hydroxybenzoate and phenol-degrading anaerobic consortium

Summary Two 4-hydroxybenzoate decarboxylase activities and a phenol carboxylase activity were found in cell-free extracts of a defined, 4-hydroxybenzoate- or phenol-grown consortium. Both decarboxylase activities were loosely membrane-associated and required K⁺ but a different pH and ion strength. Loss of activity of both decarboxylases by EDTA could be compensated by Zn²⁺ ions. The K _m values for 4-hydroxybenzoate and K⁺ of the decarboxylase activities with pH optima at 6.4 or 7.8 were 0.02 and 2.5 or 0.004 and 0.5 mm, respectively. 3,4-Dihydroxybenzoate, 3,4,5-tridydroxybenzoate, 3,5-dimethoxy-4-hydroxybenzoate and 3-chloro-4-hydroxybenzoate were also decarboxylated by both enzyme activities. The phenol carboxylase was a soluble enzyme with its pH optimum at 6.5. It required K⁺, Rb⁺ or NH _inf4 ^sup+ as monovalent, Zn²⁺, Mg²⁺, Mn²⁺ or Ni²⁺ as divalent cations and catalysed the carboxylation of phenol if 2,4-,2,3,4- or 2,4,6-hydroxybezoates were absent. The three enzyme activities were not influenced by Avidin and thus were probably not biotin-dependent enzymes. Offprint requests to: J. Winter     

Geosiphon pyriforme,an Endosymbiotic Consortium of a Fungus and a Cyanobacterium (Nostoc), Fixes Nitrogen

The bladders of Geosiphon pyriforme, an endosymbiotic consortium of a fungus and the cyanobacterium Nostoc punctiforme, show nitrogenase activity. This suggests that the organism is capable of nitrogen fixation.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Effect of chloramphenicol,antimycin A and hydroxamate on the morphogenetic development of the dimorphic ascomyceteEndomycopsis capsularis

Mitochondrial protein synthesis, primary (antimycin-sensitive) respiration and secondary (antimycin-insensitive, salicyl-hydroxamate-sensitive) respiration, have been characterized in the dimorphic yeastEndomycopsis capsularis. The inhibition by chloramphenicol (CAP) of the morphogenetic development from the yeast-like form to the mycelial structure in this yeast could represent the intervention in the morphogenetic process of mitochondrial protein synthesis, since chloramphenicol blocks in vivo and in vitro mitochondrial protein synthesis. In fact, other functions such as primary and secondary respiration, do not seem to play a role in the morphogenetic development since their inhibition by antimycin A (AA) or by salicyl-hydroxamic acid (SHAM) does not affect the process. In addition, mitochondrial protein synthesis has been shown to be uninhibited by the two respiratory inhibitors.     

Investigations on the turnover of adrenocortical mitochondria

Summary The effects of chronic administration of ACTH (up to 36 consecutive days) on the mitochondria of the zona reticularis of the rat adrenal cortex were investigated by stereologic techniques. It was found that ACTH induces two phases of hypertrophy of mitochondria alternating with two proliferative stages, which are associated with a significant decrease in the average volume of the organelles. It is suggested that, as in the zona fasciculata, ACTH controls the processes of growth and division of mitochondria in the zona reticularis. The mechanism underlying this action of ACTH as well as the differences between the responses to ACTH of the mitochondrial population of the two adrenal zones are discussed in the light of evidence indicating that mitochondria contain a complete genetic apparatus largely independent of nuclear control.The authors wish to thank Miss A. Coi and Mr. G. Gottardo for their excellent technical assistance. This work was partly supported by a contract with the CNR (C.T. 73.00663.04)     

In vitro anti-hapten response to a lactoside-conjugated protein

This paper describes the in vitro response of mouse lymphocytes to the hapten, azophenyl lactoside (Lac), coupled to the protein carrier, keyhole limpet hemocyanin, (KLH), and attempts to characterize and quantify the specific B and T cells involved. The system studied in detail has used spleen suspensions from mice which received Lac-KLH in complete adjuvant some months previously and a subsequent iv injection. The mean IgG plaque response of such cultures was about 15,000 with a corresponding IgM response about 5% of that value, similar in magnitude and Ig class distribution to in vivo responses. Primary responses were small, reflecting the low frequency of Lac precursors in normal mice. From dilution studies, we have estimated that the frequency of precursors specific for the Lac epitope in primed and normal mice is of the order of 10^?5 and 10^?7, respectively. The clonal yield of plaques from a stimulated B cell was about 150, independent of T cell concentration. The Lac response was dependent on the presence of adherent cells. It also shows a stringent requirement for carrier-specific T cells, which could not be satisfied by nonspecific T cell products or the addition of B cell mitogens. We have found that both the IgM and the IgG Lac responses are dependent on specific T cell help.     

Flux balance analysis in the production of clavulanic acid by Streptomyces clavuligerus

In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α‐ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1226–1236, 2015     

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.