Cpn60 is exclusively localized into mitochondria of rat liver and embryonic Drosophila cells

Several reports have claimed that the mitochondrial chaperonin cpn60, or a close homolog, is also present in some other subcellular compartments of the eukaryotic cell. Immunoelectron microscopy studies, using a polyclonal serum against cpn60, revealed that the protein is exclusively localized within the mitochondria of rat liver and embryonic Drosophila cells (SL2). Furthermore, no cpn60 immunoreactive material could be found within the nucleus of SL2 cells subjected to a 1 h 37°C heat-shock treatment. In contrast to these findings, immunoelectron microscopy studies, using a cpn60 monoclonal antibody, revealed mitochondrial and extramitochondrial (plasma membrane, nucleus) immunoreactive material in rat liver cells. Surprisingly, the monoclonal antibody also reacted with fixed proteins of the mature red blood cell. The monoclonal antibody, as well as cpn60 polyclonal sera, only recognize mitochondrial cpn60 in Western blots of liver proteins. Furthermore, none of the cpn60 antibodies used in this study recognized blotted proteins from rat red blood cells. Therefore, we suggest that the reported extramitochondrial localization of cpn60 in metazoan cells may be due to cross-reactivity of some of cpn60 antibodies with conformational epitopes also present in distantly related cpn60 protein homologs that are preserved during fixation procedures of the cells. © 1995 Wiley-Liss, Inc.     

Cutaneous zygomycosis caused byAbsidia corymbifera in a leukemic patient

A case of cutaneous zygomycosis caused byAbsidia coryabifera in a leukemic patient submitted to chemotherapy is reported. The lesion was located on the little finger of the right hand and probably resulted from a latent osteomyelitis. It progressed to form extensive necrotic area. No systemic infection was detected and the lesion did not appear to be associated with any trauma.     

Susceptibility of heterochromatin to aphidicolin-induced chromosomal breakage

The distribution of aphidicolin-induced chromosomal lesions was analyzed to determine the relative breakage susceptibility of euchromatin and heterochromatin in the cactus mouse, Peromyscus eremicus. The observed breakage was tested against expected distributions corresponding to the karyotypic proportions of autosomal euchromatin, autosomal heterochromatin, X-chromosomal euchromatin, and X-chromosomal heterochromatin. The distribution of induced breakage was independent of sex but dependent on the individual. In all individuals, there was a highly significant (P0.0001) deficiency in the number of breaks observed as compared to expected in autosomal heterochromatin. Sparse observations in the X chromosome and the absence of breaks in the Y chromosome precluded valid statistical tests of the sex-chromosomal distribution of induced breakage. These data indicate that the autosomal heterochromatin of Peromyscus is resistant to aphidicolin-induced chromosomal breakage and argue against a simple relationship between late replication and a general mechanism for chromosomal fragility.     

Production of hydrogen peroxide by Lactobacillus hilgardii and its effect on Leuconostoc oenos growth

In mixed culture of Lactobacillus hilgardii X₁B and Leuconostoc oenos X₂L, isolated from Argentinian wines, an amensalistic growth response was observed: Leuconostoc oenos did not grow, and after 24 h of incubation at 30°C no viable cells were detected. In pure and mixed cultures, Lactobacillus hilgardii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. The values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the action of hydrogen peroxide on the growth of Leuconostoc oenos were: 4.08g ml^-1 and 17.00 g ml^-1 respectively.     

The grass megagametophyte and its possible phylogenetic implications

The structure of the grasses megagametophyte is considered to be characteristic enough as to deserve a particular place in the megagametophyte typology. Furthermore, it is compared with those of other Monocotyledonous families to point out embryological affinities.Both are members of the Carrera del Investigador (Conicet, Argentina).     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Effects of Controlled Atmospheres on Production of Sesquiterpenoid Stress Metabolites by White Potato Tuber: Possible Involvement of Cyanide-resistant Respiration

Levels of katahdinone (solavetivone), lubimin, rishitin, and phytuberin, sesquiterpenoid stress metabolites of white potato (Solanum tuberosum), were monitored in tuber slices which were challenged with an extract of Phytophthora infestans and incubated under controlled atmospheres. A mixture of ethylene in air enhanced stress metabolite production. This enhancement was amplified by higher partial pressures of oxygen. Stress metabolite production was inhibited by salicylhydroxamic acid. These results suggest the involvement of cyanide-resistant respiration in the production of potato stress metabolites, compounds which may serve as phytoalexins.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Isolation and properties of crystalline ferredoxin from lactuca sativa

Lettuce ferredoxin has been purified to homogeneity, with a yield of 18 mg/kg of denerved leaves. It crystallizes in magnificent needles, often clustered in broom-like sheaves. The absorption spectrum showed maxima at 460, 422, 330 and 274 nm,with a ratio A₄₂₂/A₂₇₄, of 0.46. The mM absorption coefficient was 9.74 at 422 nm, and 21.62 at 274 nm. This ferredoxin showed a pI = 4.7 and an E′₀ = ?425 mV (at pH = 7.7). MWs of 12 400, 11480 and 13000 were obtained by sucrose gradient centrifugation, and on the basis of the amino acid composition and the iron content, respectively, with an average of 12 300. The amino acid analysis showed the existence of one methionine residue per mole, with 105 amino acid residues. There are two iron atoms and two labile sulfide groups per mole; 4 half-cystine residues were found by performic acid oxidation, and 5 cysteine groups when determined by titration with pHMB. The native protein is not fixed on thiol-Sepharose 4B, but it is quantitatively retained after incubation with 8 M urea. Lettuce ferredoxin showed a 62, 58 and 78% effectiveness with the spinach ferredoxin-NADP reductase, nitrite reductase and fructose-1,6-diphosphatase (FDPase), respectively, when compared with the spinach ferredoxin. This different behaviour of both ferredoxins is joined to genetic-structural relationships, and suggests that the role of ferredoxin in FDPase activation is more sophisticated than that of a mere nonspecific reductant.