Mutation analysis of the DBC2 gene in sporadic and familial breast cancer.

The expression of the recently identified tumor suppressor gene, DBC2 (Deleted in Breast Cancer 2), is frequently extinguished in breast cancer cells or tissues. Mutation analysis of the essential promoter region, all exons and exon/intron boundaries of the DBC2 gene was performed in 100 sporadic breast cancer cases by PCR-SSCP, and DHPLC, followed by direct sequencing. An additional 17 breast cancer families, who were negative for the BRCA1/2 mutations, were analyzed by direct sequencing. Three novel mutations were observed in the promoter and 5'-untranslated region (UTR) of the gene; a germ-line G>A transition in the promoter at nt -238 from the transcription start site, and two tumor-specific mutations at nt -121C>T and nt +48G>A. No deleterious mutations were detected in the coding sequence of the gene in familial and sporadic breast cancer cases. The sequence variations found within the promoter and 5'-UTR region of the gene warrant expression analysis and screening more tumor samples at this region.

Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase.

Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.

Comparative study of superoxide dismutase activity assays in Crocus sativus L. corms.

Superoxide dismutase catalyzes the breakdown of the superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods were compared for the measurement of superoxide dismutase activity in Crocus sativus L. corm extract. The methods, based on a competition between the enzyme itself and another superoxide scavenger, involved respectively cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autoxidation. Because of its accuracy, reproducibility, simplicity and cost benefit, the latter method was the most appropriate.

Thermal stability of catalases active in dormant saffron (Crocus sativus L.) corms.

Catalase activity was detected in crude extract prepared from dormant saffron (Crocus sativus L.) corms. The activity was independent of pH in the range 6.0-11.0. Thermostability studies suggested the presence of three isoenzymes with transition temperatures of 30 degrees C, 45 degrees C and 60 degrees C, respectively, as given by Arrhenius plots. When stained for catalase activity gel electropherograms of extract revealed 3 distinct bands with apparent molecular weight of 323,000, 295,000 and 268,000, respectively. Thus it appeared that at least three isoenzymes of catalase were present in dormant saffron corms.

The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium.

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.

Kinetics of peroxidases in guinea pig bone marrow under immunostimulation.

Eosinophil peroxidase and myeloperoxidase play an important role in the host defense. Both enzymes are present in bone marrow, synthesized by blood progenitor cells. This research investigated the kinetic properties of peroxidases under immunostimulation in guinea pig bone marrow. Results suggest that there are at least two myeloperoxidase isozymes and at least three eosinophil peroxidase isozymes in guinea pig bone marrow and that some of these isozymes are expressed upon immunostimulation.

DNA-mobility shift assay and the detection of anti-DNA IgG in systemic lupus erythematosus patients.

Although the presence of antibodies against double-stranded (ds) DNA is highly specific of systemic lupus erythematosus (SLE), it is not detected in all SLE patients, perhaps due to a lack of sensitivity of the tests routinely used to assay anti-(ds) DNA. Looking for an alternative assay, this study explored the applicability of a DNA-mobility shift assay for the detection of anti-(ds) DNA; furthermore, the study compared the use of Salmonella typhimurium DNA with that of calf thymus DNA in the assay. After electrophoresis, samples containing S. typhimurium DNA and IgG from SLE sera showed marked alterations in DNA electrophoretic mobility when compared to DNA alone. In our sampling, SLE patients who tested negative for anti-(ds) DNA antibodies with routinely used assays such as Crithidia luciliae immunofluorescence test, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), tested positive for anti-(ds) DNA with the DNA mobility shift assay using S. typhimurium DNA. Incubation with IgG from control sera in the same proportions as above did not affect S. typhimurium DNA electrophoretic mobility. When S. typhimurium DNA was replaced by calf thymus DNA, the effect on the DNA mobility was less pronounced and less reliable. These results indicated that a DNA-mobility shift assay would be a useful alternative for the unequivocal detection of abnormal titers of anti-(ds) DNA antibodies. Furthermore, data indicated a greater ability of the IgG from SLE patients to form complexes with S. typhimurium DNA than with calf thymus DNA, suggesting an alternative testing DNA which may lead to a more sensitive anti-(ds) DNA detection.

Morphological basis for multiple interactions of ethidium bromide (EB) with yeast Candida utilis.

Exposure of yeast cells to EB produced multiple effects on the cellular organelles: changes in the plasma membrane characterized by 75 to 110 nm deep pits; polymorphisms of the mitochondria ranging from cup-shaped, ring-shaped, ribbon-shaped, dumbbell-shaped structures to finally the formation of very elongated mitochondria (up to 4.5 micron in length); an increase in the length and number of endoplasmic reticulum; an increase in the number of cytoplasmic vesicles whose diameter varied between 25 to 45 nm. Furthermore, EB inhibited cytochrome c oxidase and cytochrome b biosynthesis, stimulated cytochrome c biosynthesis and uncoupled oxidative phosphorylation.

The surfactant system of the lung in the Mexican axolotl Ambystoma mexicanum.

The broncho-alveolar space of axolotl contains numerous osmiophilic structures, which have been classified morphologically into 3 types of inclusions. Type I inclusions exhibited lattices of square to rectangular grid patterns with membranous elements 6 nm thick. This lattice was crossed by a 2 nm dense line. Type II inclusions were composed of 7 nm dark lines and 15.5 nm light lines in an alternating repeating pattern. Furthermore, the light lines showed an intrapenoid line of 2 nm. Type III inclusions consisted of concentric whorls of lamellae similar in form to spider webs. Only type III inclusions were identified in grand alveolar cells.

Biochemical characterization of the OXI mutants of the yeast Saccharomyces cerevisiae.

OXI mutants in Saccharomyces cerevisiae lack a functional cytochrome c oxidase. Wild type and OXI mutants were grown in the presence of radioactive delta-amino[14C]levulinic acid, a precursor of porphyrin and heme, and [3H]mevalonic acid, a precursor of the alkyl side-chain of heme a. SDS polyacrylamide gel electrophoresis of the delipidated mitochondria showed that delta-amino[14C]levulinic acid was distributed into three bands migrating in the regions of Mr 28 000, 13 500, and 10 000, while [3H]mevalonic acid was found in a single band with apparent Mr of 10 000. The immunoprecipitates obtained by incubating the solubilized mitochondria of any OXI mutant with antibodies against cytochrome c oxidase, showed, after delipidation, a high specific radioactivity due to delta-amino[14C]levulinic acid and [3H]mevalonic acid. This suggested that a prophyrin a was present in all these OXI mutants. HCl fractionation confirmed the presence of porphyrin a in the apooxidase of these mutants. Atomic absorption spectra of the immunoprecipitate of cytochrome c oxidase showed that copper was not detectable in the mutant OXI IIIa which lacked subunit 1, but was present in the mutant OXI IIIb, which exhibited a minor alteration in the electrophoretic mobility of subunit 1. In OXI I and II mutants there was a 50% reduction in the amount of copper in the immunoprecipitated cytochrome c oxidase. These observations may be interpretable as follows: (1) alterations in polypeptide biosynthesis due to the OXI mutations lead to an improper configuration of cytochrome c oxidase, so that ferrochelatase cannot transfer iron into porphyrin a; (2) subunit I is the binding site for copper, but the mutations in subunits II and III alter the binding site of one of the two copper atoms in subunit I.

Mouse megakaryocytes secrete acetylcholinesterase.

Acetylcholinesterase (AchE), an essentially specific and early marker of rodent thrombocytic series, in several tissues acts both as a constituent of the cellular membrane and as a secretory enzyme. This study presents the ultrastructural transport and fate of this substance in the megakaryocytes of mouse bone marrow, using Tranum-Jensen and Behnke's adaptation of the indirect thiocholine method. It is shown that megakaryoblasts and megakaryocytes undergo a typical secretory cycle consisting of (1) enzyme synthesis and segregation on the endoplasmic reticulum and nuclear envelope, (2) enzyme concentration in AchE-vesicles and AchE-granules formed from the cisternae of the Golgi apparatus, and (3) discharge in the demarcation membrane system and extracellular space. The AchE-vesicles and granules appear to be hitherto unrecognized megakaryocytic organelles as they do not resemble alpha nor the dense granules, and their mode of formation and fate differ from those of primary lysosomes and peroxidase granules. Released platelets reveal AchE activity in the open canalicular system. The data are compatible with the hypothesis that by controlling acetylcholine concentration in hematopoietic tissues, the secretion of AchE by megakaryocytes can modulate the proliferative activity of megakaryocytes progenitors.

Acetylcholinesterase in mammalian erythroid cells.

The biosynthesis of acetylcholinesterase in mammalian erythroid cells during differentiation and maturation was studied using a cytochemical method. Acetylcholinesterase was actively synthesized in basophilic erythroblasts I and II, and polychromatophilic erythroblasts, where it was present in the nuclear membrane, endoplasmic reticulum and Golgi apparatus. In orthochromatophilic erythroblasts the enzyme was present only in the Golgi apparatus, thus suggesting the end of the biosynthesis of acetylcholinesterase at this stage of maturation. In mouse and human marrow, evidence was also found suggesting the secretion of acetylcholinesterase into the extracellular compartment. No acetylcholinesterase reaction product was found in cat erythroblasts.

[Secretion of acetylcholinesterase by megakaryocytes in mice]. / La sécrétion d' acétylcholinestérase par les mégaryocytes de souris.

Acetylcholinesterase, an early and nearly specific marker of Rodent thrombocytic cells, undergoes a typical secretory cycle leading to the release of the enzyme in the demarcation system and the outer cellular space of bone marrow megakaryocytes. This secretion process suggests that megakaryocytes may adapt the amplification of their own precursors by modulating the concentration in cholinergic compounds in hematopoietic tissues.

Mitochondrial morphology in the spermatozoa of the Mexican axolotl, Ambystoma mexicanum.

Thin-section and freeze-fracture electron microscopy of immature and mature spermatozoa of the Mexican axolotl, Ambystoma mexicanum, revealed numerous small spherical mitochondria with diameters ranging from 0.15 to 0.22 micrometers. Both the spherical form and the small size of these mitochondria were confirmed by serial thin-section studies. In mature spermatozoa, the mitochondria are located in the midpiece region, in tight contact with each other, exhibiting an almost crystalline arrangement. They do not surround the midpiece, but form a semicircular sheet over the sustained filament. The portion of the midpiece on the side of the undulating membrane and the flagellum is devoid of mitochondria. The plasma membrane in the midpiece region is tightly apposed to the mitochondria, so that in freeze-fracture or scanning electron microscopy the mitochondria seem to protrude through the plasma membrane. We suggest that the unusual organization of mitochondria in axoloti sperm facilitates the oxidative processes and increases the efficiency of ATP production and/or distribution within the cell.

Acetylcholinesterase in cat megakaryocyte. Evidence for extracellular secretion.

Cytochemical localization of acetylcholinesterase (AchE, E.C.3.1.1.7) was studied in cat bone marrow. The reaction product precipitate was present only in megakaryocytes, and all other marrow cells were AchE-negative. Moderate electron dense AchE reaction product was found in basophilic megakaryocytes in the nuclear membrane, endoplasmic reticulum, Golgi complex, and demarcation membrane system (DMS). In granular and thrombocytogenic stages the intense AchE reaction product was present in all cytoplasmic membranes. The Golgi-derived AchE-positive granules were found to fuse with DMS and their contents were discharged into the extracellular compartment.

Identification of porphyrin present in apo-cytochrome C oxidase of copper-deficient yeast cells.

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [3H]leucine and delta-amino[14C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [3H]leucine associated with six bands and delta-amino[14C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with delta amino[14C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thin-layer chromatography of the radioactivity found in 20% HCl showed an RF value identical to that of purified porphyrin a. When delta-amino[3H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells was converted into heme a, and this conversion was prevented by cycloheximidine. These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficiency.

Observations on the mitochondrial reticulum in the yeast Candida utilis as revealed by freeze-fracture electron microscopy.

Freeze-fracture of Candida utilis yeast cells grown to early logarithmic phase (5 h) and stationary phase (24 h) revealed a branched mitochondrial reticulum both in longitudinal and transverse cross-fracture studies. In contrast, the longitudinal and transverse sections of permanganate-fixed cells showed that the small ovoid or spherical mitochondria were located at the periphery of the cell, without formation of a reticulum. Data indicate that the separate mitochondrial profiles seen in thin sections of permanganate-fixed yeast cells are not separate round or ovoid mitochondria, but rather are cross-sections of the mitochondrial reticulum.