Mechanism of sodium independent calcium efflux from rat liver mitochondria

On the basis of primarily two types of observations, it has been suggested that the Na+-independent Ca2+ efflux mechanism of rat liver mitochondria is a passive Ca2+-2H+ exchanger. First, when a pulse of acid is added to a suspension of mitochondria loaded with Ca2+, a pulse of intramitochondrial Ca2+ is often released, even in the presence of the inhibitor of mitochondrial Ca2+ influx, ruthenium red. Second, at a pH near 7, the stoichiometry of Ca2+ released to H+ taken up by Ca2+-loaded mitochondria, following treatment with ruthenium red, has been observed to be 1:2. This evidence for a Ca2+-2H+ exchanger is reexamined here by studying the release of Ca2+ upon acidification of the medium by addition of buffer, the dependence of liver mitochondrial Ca2+ efflux on external medium pH and intramitochondrial pH, and the Ca2+-Ca2+ exchange properties of the Ca2+ efflux mechanism. These studies show no pulse of mitochondrial Ca2+ efflux when pH is abruptly lowered by addition of buffer. The stoichiometry between Ca2+ and H+ fluxes is found to be highly pH dependent. The reported 1:2 stoichiometry between Ca2+ efflux and H+ influx is only observed at one pH. Furthermore, the rate of Ca2+ efflux from mitochondria is found to increase only very slightly at most as suspension pH is decreased. The rate of Ca2+ efflux is not found to increase with increasing intramitochondrial pH. Finally, no Ca2+-Ca2+ isotope exchange can be demonstrated over the Na+-independent efflux mechanism (i.e., in the presence of ruthenium red). It is concluded that these data do not support the hypothesis that the Na+-independent Ca2+ efflux mechanism is a passive Ca2+-2H+ exchanger.     

Maternal effects and temperature-sensitive period of mutations affecting embryogenesis in Caenorhabditis elegans

We have used standard tests to investigate the nature of gene expression of a new set of temperature-sensitive mutants defining 30 emb genes (essential for embryogenesis) in the nematode Caenorhabditis elegans. The mode of gene expression as determined by progeny tests for parental effects divides the genes into four classes. For 18 genes maternal gene expression is necessary and sufficient for normal embryogenesis; for 2 genes zygotic expression is necessary and sufficient; for 7 genes either maternal or zygotic expression is sufficient; for 3 genes both maternal and zygotic expression are necessary. One mutant displayed partial paternal sufficiency. The results of temperature-shift experiments define two “execution stages,” corresponding to the limits of the temperature-sensitive period (TSP), and indicate the nature and the time of action or synthesis of the gene products. Most of the maternally expressed genes have very early execution stages indicating translation before fertilization, but some are temperature sensitive late in embryogenesis. Early execution stages for 2 zygotically necessary genes demonstrate that the zygotic genome can be active in the earliest stages of embryogenesis. All taken together, the mode of gene expression, TSP, and arrest stage (terminal phenotype) allow us to classify functionally and begin to order the genes essential for embryogenesis. The results indicate a preeminent role for maternal genes and gene products in embryogenesis, in agreement with the results of others.     

Some novel flavin-nicotinamide biscoenzymes and their reactivities

The present study was undertaken to investigate flavin-nicotinamide reactions and interactions. A series of novel flavin-nicotinamide biscoenzymes have been synthesized by a general three-step procedure. The structures of these compounds were confirmed by nuclear magnetic resonance spectra, absorption spectra and elemental analysis. These compounds consist of short linear hydrocarbon chains interconnecting the N-1 of nicotinamide and the N-10 of the 7,8-dimethyl-isoalloxazine ring. The compounds were reduced with sodium dithionite (Na₂S₂O₄) and the flavin portion was reoxidized with ferricyanide. Re-reduction of the flavin portion by the nicotinamide portion of the molecule was followed anaerobically at 442 nm. When the interconnecting hydrocarbon chain was unsaturated, a second order reaction was observed with a rate equal to that of lumiflavin and 1-propyl-1,4-dihydronicotinamide (NprNicH₂) under the same conditions. When the two halves of the biscoenzymes were connected by saturated three- and four-carbon chains, the expected unimolecular reaction was not observed. Instead, the reduced biscoenzyme, after separation from excess sodium dithionite, was shown to have a strong absorption at 298 nm. This absorption is characteristic of hydration of dihydronicotinamides at the 5,6-double bond.In further studies, the C₃-biscoenzyme exhibited an absorption at 600 nm due to a complex between the reduced flavin and oxidized nicotinamide portions of the molecule. Absorbance at 600 nm increased linearly with the C₃-biscoenzyme concentration, clearly indicating that this is an intramolecular complex. When the C₃-biscoenzyme was at 0°C in 60–75% dimethylformamide buffer solution, no absorption at 600 nm was observed. When excess dithionite was removed, the spectrum under these conditions showed definite peaks at 297 and 357 nm. These respective peaks were attributed to hydrated dihydronicotinamide and dihydronicotinamide species present in the reaction mixture.The reduced flavin was postulated to be a catalyst for the hydration of dihydronicotinamide. This hypothesis was tested by incubating 1-propyl-1,4-dihydronicotinamide alone and with several concentrations of reduced riboflavin under basic anaerobic conditions. The results show that the reduced flavin increases the rate of disappearance of the dihydronicotinamide species and that the product shows an absorption near 298 nm. These results indicate that a reduced form of the flavin nucleus catalyzes the hydration of dihydronicotinamides.     

STUDY OF MITOCHONDRIA IN RAT LIVER : Quantitative Electron Microscopy

The electron microscope has been used to determine the weight distribution of isolated subcellular particles from normal rat liver. The following results are reported: (1) There exist at least two well defined weight populations of subcellular particles; their respective median weights are 1.3 x 10^-14 and 11 x 10^-14 gm. The lighter fraction is considered to consist of lysosomes, the heavier of mitochondria. (2) The mitochondrial fraction shows a log-normal distribution of the particle weight. (3) By the introduction of morphologic criteria, the mitochondrial fraction is divided into two groups, one consisting of a spherical, the other of an oblong type of particle. The data found support the following concepts: (a) Mitochondria increase their weight from a certain size up by linear growth. (b) Mitochondria divide. The division is not necessarily symmetric; in all cases, however, one part of the division product is a spherical particle. It is felt that these results constitute a valuable demonstration of the general capabilities of quantitative electron microscopy and may stimulate many other useful applications of this technique in cytology, bacteriology, and virology.     

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Summary The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of -glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous -glucosidase. End-product inhibition of the cellulase and -glucosidase can be more effectively reduced by employing a CHF process than by supplemental -glucosidase.Offprint requests to: C. M. Hogan     

Soil pCO2, soil respiration,and root activity in CO2-fumigated and nitrogen-fertilized ponderosa pine

The purpose of this paper is to describe the effects of CO₂ and N treatments on soil pCO₂, calculated CO₂ efflux, root biomass and soil carbon in open-top chambers planted with Pinus ponderosa seedlings. Based upon the literature, it was hypothesized that both elevated CO₂ and N would cause increased root biomass which would in turn cause increases in both total soil CO₂ efflux and microbial respiration. This hypothesis was only supported in part: both CO₂ and N treatments caused significant increases in root biomass, soil pCO₂, and calculated CO₂ efflux, but there were no differences in soil microbial respiration measured in the laboratory. Both correlative and quantitative comparisons of CO₂ efflux rates indicated that microbial respiration contributes little to total soil CO₂ efflux in the field. Measurements of soil pCO₂ and calculated CO₂ efflux provided inexpensive, non-invasive, and relatively sensitive indices of belowground response to CO₂ and N treatments.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits