Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli

We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.     

Novel peripheral blood-derived human cell lines with properties of megakaryocytes

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.     

Uptake of transferrin and iron by cultured rat placental cells

This paper describes a method for the culture of rat placental cells. The method involved separation of the basal layer from the labyrinth and sequential digestion of the cells. The cells were demonstrated not to be fibroblasts and are described in terms of their appearance under the light and electron microscopes. Transferrin and iron uptake by the cells was examined and compared with results achieved using other methods of study. The results showed that transferrin bound to receptors on the cell surface and that the transferrin, once bound, was taken into the cell. Only this internalized transferrin was capable of donating iron to the cells. The iron was accumulated within the cells and did not appear to be released to the incubation medium. The apparent dissociation constant (Ka) for transferrin was found to be 6.96 X 10(6) M-1, a value similar to that described by earlier workers. The placental cells had 3.4 X 10(11) binding sites/microgram DNA, equivalent to approximately 1 X 10(6) sites/cell. From these data, and from the rate of accumulation of iron by the cells, the receptor turnover time was estimated as being between 5 and 10 min.     

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs.     

Hemopexin-mediated heme transport to the liver. Evidence for a heme-binding protein in liver plasma membranes

Isolated liver plasma membranes interact with heme-hemopexin and effect the removal of heme from the complex. This heme is rapidly accumulated by a previously undescribed heme-binding membrane component (HBC). This intrinsic membrane component can be solubilized from the membrane with Triton X-100 in a form that retains the ability to bind heme. Solubilized HBC was shown to be distinct from hemopexin itself, free heme, ligandin, globin, heme oxygenase, cytochrome P-450, and albumin. Since formation of the heme-HBC complex is effected by the interaction of heme-hemopexin with its receptor, HBC may either be a subunit of the heme-hemopexin receptor or a separate protein that interacts with the receptor. HBC can also bind heme (Kd apparent 200 nM) that is presented to it in a nonprotein bound form, showing true heme-binding activity. HBC is proteinaceous since treatment with proteases, heat, and disulfide bond reducing agents diminishes its ability to bind heme. HBC and any associated detergent elutes from Sephacryl S-200 with an apparent molecular weight of 115,000 and Stokes radius of 7.5 nm. This component, which may comprise 0.5% of liver plasma membrane protein, appears to have an acidic pI since it adsorbs to DEAE-cellulose at pH 7.4 but not to CM-cellulose at pH 6.4. In sucrose gradients, HBC migrates with S values of 1.69 and 4.02, suggesting that it has subunits or that it forms multimers under these conditions.     

Thermophilic anaerobic spirochetes in New Zealand hot springs

Abstract Electron and light microscopy revealed the presence of spirochetes in New Zealand thermal springs. The spirochete population in one spring studied (Kuirau Lake) was affected by fluctuations in temperature and/or pool level. A pure culture of the strictly anaerobic bacterium revealed that it grew optimally at a temperature of 45–50°C, with no growth occurring above 60°C, and a pH of 7.0–7.5 with no growth occurring at pH 5.5 or 8.5. Growth was inhibited by chloramphenicol, penicillin, streptomycin, tetracycline and neomycin but not by d -cycloserine, novobiocin or phosphomycin at 10 μg/ml. A wide range of carbohydrates were utilized but not organic acids. Acetate was the major end product of glucose fermentation with substantial amounts of ethanol and traces of lactate being produced.     

Comparison of a Thermoanaerobium sp. from a New Zealand hot spring with Thermoanaerobium brockii

Abstract An anaerobic ethanologenic strain of extremely thermophilic bacteria isolated from a New Zealand hot spring resembled Thermoanaerobium brockii in morphology and cell-wall ultrastructure. However, antibodies produced against the New Zealand isolate did not crossreact with the type strain of T. brockii . The New Zealand isolate strain Tok6-B1 fermented a wider range of carbohydrate substrates, including pentoses, and was less inhibited by a hydrogen atmosphere. Ethanol and acetate were major end-products and lactate a minor product of glucose fermentation. Under a hydrogen atmosphere, these 3 end-products were formed in approximately equal amounts.