Myoglobin and cytochrome b5: a nuclear magnetic resonance study of a highly dynamic protein complex

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b(5) titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a K(a) in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b(5), with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.     

Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Isolation and Characterization of Nisin-Resistant Leuconostoc mesenteroides for Use in Cabbage Fermentations

Leuconostoc mesenteroides strains that are resistant to high levels of nisin (up to 25,000 IU/ml in broth) were isolated. These nisin-resistant mutants were evaluated to determine their potential use as starter culture strains for cabbage fermentations. We found that some L. mesenteroides strains could be adapted to high levels of nisin resistance, while others could not. The nisin resistance trait was found to be stable for at least 35 generations, in the absence of nisin selection, for all mutants tested. The effects of nisin and salt, separately and in combination, on growth kinetics of the nisin-resistant strains were determined. Salt was the most influential factor on the specific growth rates of the mutants, and no synergistic effect between nisin and salt on specific growth rates was observed. The nisin-resistant strains were unimpaired in their ability to rapidly produce normal heterolactic fermentation end products. The use of these L. mesenteroides mutants as starter cultures in combination with nisin may extend the heterolactic phase of cabbage fermentations.     

Direct detection of rhizosphere-colonizing Pseudomonas sp. using an Escherichia coli rRNA promoter in a Tn7-lux system

Abstract A promoterless Tn7- lux system conferring bioluminescence was fused with an Escherichia coli rRNA gene promoter and compared with neo - or lac-luxCDABE analogs after introduction in Pseudomonas cells. Fusion of the ribosomal promoter with luxCDABE genes increased the bioluminescence of cells by approx. 100- to 1500-fold over the neo-lux system depending on the growth conditions and bacterial strain. When the cells were grown in suspension culture, light production and growth were strongly dependent on the nutrient composition of the medium. Root-colonizing competence was tested in nonsterile soil by autophotographic detection of bacterial bioluminescence on plant roots. The lower detection limit of the autophotographic method for roots inoculated with Pseudomonas fluorescens 2–79 was 10⁵ cfu g⁻¹ fresh root weight. The new bioluminescence marker did not require addition of supplemental nutrients or the aldehyde substrate for the luciferase enzyme and provides a simple and highly sensitive detection method for long term in situ studies on the microbial ecology of specific bacterial strains.     

Binding sites for 3H-LTC4 in membranes from guinea pig ileal longitudinal muscle

Leukotriene (LTC₄) is one of the components of Slow Reacting Substance of Anaphylaxis (SRS-A) and is a potent constrictor of guinea pig ilea. The contraction is likely to be a receptor-mediated process. Here we report the existence of specific binding sites for ³H-LTC₄ in a crude membrane preparation from guinea pig ileal longitudinal muscle.At 4°C in the presence of 20 mM Serine-borate, binding increases linearly with protein concentration, reaches equilibrium in 10 minutes, and is reversible upon addition of 3 × 10^?5M unlabelled LTC₄. The dissociation curve is consistent with the existence of more than one class of binding site. Ca⁺⁺ and Mg⁺⁺ greatly enhance the binding of ³H-LTC₄ at equilibrium. In the presence of 5mM CaCl₂ and MgCl₂ not only LTC₄ (IC₅₀ 10^?7M), but also LTD₄ (albeit with much lower affinity, IC₅₀ = 6 × 10^?5M) and the SRS-A antagonist FPL 55712 (IC₅₀ = 10^?5M) can compete with ³H-LTC₄ for its binding sites. FPL 55712 only displaces 60–70% of the total amount bound, while LTC₄ displaces 90–95%.These studies indicate that multiple classes of binding sites exist for ³H-LTC₄ in guinea pig ileal longitudinal muscle, and that at least part of these binding sites might be related to the ability of LTC₄ to contract guinea pig ilea.     

Effective size and the persistence of ecosystems

Summary Ecosystems distributed in space have an effective size, reflecting both their absolute size (extent) and their fine-scale physical structure (viscosity). In this paper, a general mathematical model of a predator-prey interaction is presented via the phase-plane graphs of Rosenzweig and MacArthur (1963) to show one reason why ecosystems of larger effective size should persist longer than smaller ones: oscillations of population densities tend to be displaced farther from extinction thresholds—even in spatially homogeneous systems. Experimental results obtained by Gause and Luckinbill with protozoa and Huffaker with mites are interpreted in this context.     

Immunologic function during adjuvant BCG immunotherapy for malignant melanoma: Induction of anergy

Summary Serial tests of immunological function were performed on 28 patients participating in a randomized controlled clinical trial of adjuvant Tice-stain BCG immunotherapy administered by tine technique for malignant melanoma. Cryopreserved lymphocyte samples obtained prior to study entry and at 3 and 6 months there-after were tested by mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML), antibody-dependent cell-mediated cytotoxicity (K cell), and natural killing (NK cell) assays, the last two assays being performed with the Chang cell line. Delayed-type hypersensitivity (DTH) skin tests to recall antigens were performed at the same intervals.At entry to the study in vitro lymphocyte reactivity of patients was similar to that of normal controls, and most (75%) of the patients reacted to at least one recall antigen. Serial lymphocyte reactivity measured by the in vitro tests was not different in the BCG and control groups, but BCG treatment was associated with a marked, statistically significant (P<0.01) reduction in DTH skin test reactivity. BCG therapy was not shown to delay recurrence of the disease.     

(51CR) sodium chromate incorporation ito the soluble protein fractin of the human erythrocyte: binding not associated with the hemoglobin monomeric subunit.

The distribution of radioactive chromium on proteins isolated in the soluble fraction of the human erythrocyte was studied utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The radioactive label was associated with the hemoglobin monomeric subunit and, unexpectedly, with another soluble fraction protein. This protein was approximately 6% of the total isolated protein, contained approximately 20% of the radioactivity, and had an estimated molecular weight of 26,700 daltons.