Cytotoxicity of cholesterol oxidase to cells of hypercholesterolemic guinea pigs

1. A high cholesterol diet caused guinea pig erythrocytes to become sensitive to lysis by cholesterol oxidase (CO), a protein not hemolytic to normal cells. 2. Lysis was associated with conversion of membrane cholesterol to its oxidation product (delta-4-cholesten-3-one). 3. Intravenous injection of CO to hypercholesterolemic guinea pigs produced a reduction in serum cholesterol, but was not lethal as it was in rabbits. 4. Homogenized spleen, liver and kidney from the hyperlipidemic animals were sensitive to in vitro cholesterol oxidation while tissues from non-lipemic animals were resistant to modification.     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.     

Immunohistochemical localization of cytochrome P450 3A in human pulmonary carcinomas and normal bronchial tissue

The cytochrome P450 (CYP) enzymes metabolize drugs and other xenobiotics in liver and also in some extrahepatic tissues. We have studied the expression and localization of CYP3A in primary lung tumours and normal lung tissue from the same patients. Thirtytwo patients undergoing partial or total lung resection for therapy of primary pulmonary carcinoma were included in this study. Immunohistochemical staining for CYP3A was performed with a modification of the ABC technique. Eight of the 32 cases of primary pulmonary carcinoma showed expression of CYP3A. In 12 of the 32 cases of normal tissue, the seromucous glands were positive for CYP3A. The bronchial epithelium was positive for CYP3A in 11 cases. We observed no correlation between CYP3A expression in tumour tissue and that in seromucous glands or bronchial epithelium. We conclude that CYP3A is present in both normal and cancerous lung tissue. Our findings suggest, however, no co-expression of CYP3A in lung cancer.     

Cytomorphology of primary small-cell (Merkel-cell) carcinoma of the skin in fine needle aspirates

The histologic appearance of primary small-cell carcinoma of the skin (the so-called Merkel-cell tumor) is similar to other small-cell tumors that may metastasize to the dermis. Significance has been placed on the electron microscopic appearance of this tumor since the ultrastructural features of this neoplasm are helpful in distinguishing it from most of the other neoplasms considered in the differential diagnosis. To determine whether any additional morphologic criteria might exist to distinguish this neoplasm, the fine needle aspirate appearance of a primary small-cell carcinoma of the skin was studied and compared to that of similar preparations of other small-cell tumors that could potentially involve the dermis. Cells of this unusual tumor were round and showed neither cohesiveness nor nuclear molding. Mitoses were numerous. The chromatin pattern was bland. The cytologic features of this tumor can aid in the distinction of primary small-cell carcinoma of the skin from other metastatic small-cell neoplastic lesions in the dermis of adults.     

Glutamate mutase from Clostridium cochlearium. Purification, cobamide content and stereospecific inhibitors.

Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260-3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-[alpha-(aden-9-yl)]-cob(II)amide. A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy. A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis. Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM). (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.     

Copper regulation of ceruloplasmin in copper-deficient rats.

In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.     

Effect on sphingomyelin-containing liposomes of phospholipase D from Corynebacterium ovis and the cytolysin from Stoichactis helianthus

The toxic, sphingomyelin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from Corynebacterium ovis was purified to near homogeneity. It has a molecular weight of 31 000 and a pI of approx. 9.8. Although not cytolytic itself, it protected red cells from hemolysis by staphylococcal sphingomyelinase (beta-hemolysin) and helianthus toxin. The apparently non-enzymatic cytolysin (helianthus toxin) from the sea anemone Stoichactis helianthus also interacts with membrane sphingomyelin. C. ovis and helianthus toxins were compared with regard to their effects on liposome model membranes, and they were found both to produce changes analogous to those in erythrocytes. Only helianthus toxin caused release of trapped glucose marker, but liposomes could be protected from release by pretreatment with C. ovis toxin. Both toxins demonstrated binding to sphingomyelin-containing liposomes, but only the bacterial sphingomyelinase catalyzed the release of choline from these vesicles.