Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

On the transposition of copia-like nomadic elements in cultured Drosophila cells

We studied the stability of the genomic distribution of six retrotransposon families in long-term and short-term cultures of Drosophila cells. In a subclone derived from Kc cells, no significant rearrangements were detected over an 8 year period. On the contrary, extensive reshuffling and amplification of transposon families were observed in recently established cell lines. These results show that in cultured Drosophila cells transposition appears to be restricted to the transition from the embryo to continuous cell lines.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.     

A NEW GENUS OF PROTOSTELIDS SHOWING AFFINITIES WITH CERATIOMYXA

A new genus and species of the Protosteliida (Mycetozoa), Ceratiomyxella tahitiensis, was isolated from dead plant material—var. tahitiensis from Tahiti and var. neotropicalis from Brazil and Colombia. The sporocarps have deciduous spores borne singly on slender hollow stalks; zoocysts with anteriorly flagellate planonts are produced. The trophic stage is comprised of uninucleate to plurinucleate amoeboid cells and reticulate plasmodia; the uninucleate cells become flagellate in water. The prespore cells and spores are plurinucleate. Sexuality has not been demonstrated. Var. tahitiensis has globose spores and produces its zoocysts just after spore germination, whereas var. neotropicalis has subglobose spores and forms zoocysts later in the life cycle. The species is thought to show phylogenetic relationships with Ceratiomyxa, which was recently transferred to the Protosteliida by Olive.     

MONOGRAPH OF THE GENUS PROTOSTELIUM

Three new species of Protostelium (Order Protostelida of the Mycetozoa) are described: P. irregularis, P. zonatura, and P. pyriformis, all with rather wide distribution and occurring on dead attached plant parts, less often in soil and humus. The latter two species differ from the first and from the other two known species, P. mycophaga Olive and Stoianovitch and P. arachisporum Olive, in the endogenous origin of the stalk. P. mycophaga var. crassipes, with vesicular stalk bases, is the third variety of that species to be described. A key to the genus is included.     

An electron microscopical study of developing sympathetic neurons in man

Summary An electron microscopic study has been made of the sympathetic ganglia of a 15 and a 17 week old male human fetus. The fetal sympathetic neurons were densely packed in a scanty connective tissue matrix which also contained blood vessels. The fetal sympathetic neurons had a large, electron-light nucleus with one or two nucleoli, and was of a somewhat mottled appearance due to irregularly dispersed aggregates of fine and coarse granules. The perikaryon usually formed a thin envelope around the nucleus and contained, except for large pigment granules, all intracytoplasmic structures which were also found in mature sympathetic neurons. Adjacent sympathetic cells were either in immediate contact, or slightly separated by a wedge of electron-light satellite expansions, or lined by primitive axons. The satellite cells were in the early state of development. Electron-dense axons either stood side by side with, or were slightly engulfed by light Schwann cell expansions and formed distinct bundles surrounded by a common basement membrane. There was practically no trace of myelin formation or Schwann cell wrapping characteristic for unmyelinated fibers as seen in the adult.This investigation was supported (in whole) by United States Public Health Service Grant NB-01879-05, Institute for Nervous Diseases and Blindness.Grateful acknowledgment is made to Professor Dr. John Lind who madea vailable the fetal material through the Laboratory of Prenatal Growth and Development, Karolinska Institutet, Stockholm, Sweden.The authors wish to thank Docent Dr. Gunnar Bloom who provided the facilities necessary to prepare the fetal material for electron microscopical examination, in his laboratory for Cell Research, Karolinska Institutet, Stockholm, Sweden.