Divalent cation binding to ceruloplasmin

Binding of calcium to human and sheep ceruloplasniin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The K _d for calcium was found to be 1.4mm. Magnesium also bound to ceruloplasmin, with K _d = 0.3 and 0.7 mm for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the T _m value, from 73.8 to 83.1°C, was observed for sheep ceruloplasmin in the presence of calcium. The T _m value of the human protein was only slightly altered by calcium (from 85.1 to 87°C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.     

INTERACTIONS BETWEEN SOUTH AFRICAN FUR SEALS and THE PURSE-SEINE FISHERY

Operational interactions occur between South African (Cape) fur seals and the purse-seine fishery in South Africa. Seals eat fish from the nets but the main concern is the activity of seals causing fish to sound, resulting in loss, or partial loss of the catch. Detectable loss occurs on approximately 5% of hauls. In addition to this, smaller quantities may be lost on each haul as seals depress the net float line while moving into and out of the net. The cost of seal interference is difficult to quantify because the mass of fish lost is not known, and the loss is essentially one of additional fishing time required to fill the total allowable catch for the fishery. An estimate of the cost is calculated as between 1.6% and 4.1% of the landed value of the fishery. Some seals are killed by fishermen when they pose a potential threat to the safety of the crew aboard, and some may drown if caught in the net or pump. Overall this is probably fewer than a thousand seals per year. It is also known that if seals are thought to be disturbing the fish, these seals near the nets may be deliberately killed by purse-seine fishermen. Overall this mortality, whether deliberate or incidental, is probably negligible in terms of the population size of South African fur seals.     

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin

In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.     

Highly transformable mutants of Streptomyces fradiae defective in several restriction systems

Summary Streptomyces fradiae JS85 is a mutant defective in tylosin production and an efficient recipient for conjugal transfer of tylosin genes. JS85 was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and derivatives defective in restriction were isolated by sequential selection for increased transformability by several plasmid DNAs. From the number of mutation and selection cycles required to eliminate most restriction, it was estimated that wild type S. fradiae expressed at least five restriction systems. From the patterns of restriction enzyme digestion of chromosomal DNA observed in the series of mutants that became progressively less restricting, it was suggested that wild type S. fradiae normally expresses modification (and presumably restriction) systems similar or analogous to PstI, XhoI, ScaI and EcoRI. The least restricting mutant of S. fradiae was readily transformable by many plasmids, including a bifunctional cosmid vector containing a large insert of Streptomyces DNA.     

High frequency conjugal transfer of tylosin genes and amplifiable DNA in Streptomyces fradiae

Summary Genes encoding enzymes for tylosin biosynthesis, genes involved in the expression of resistance to tylosin (Tyl), hygromycin B (Hm), chloramphenicol (Cm), and mitomycin C (MC), and a single copy of an amplifiable unit of DNA (AUD) were jointly transferred at very high frequencies by conjugation from several different Streptomyces fradiae strains to S. fradiae JS85, a mutant defective in many or possibly all tylosin biosynthetic reactions and containing a multiple tandem reiteration of the AUD. No recombination was observed between nar, rif and spc genes in conjugal matings, but recombination was observed between these genes after protoplast fusion. Tylosin biosynthetic genes were transferred at a much lower frequency to S. fradiae JS87, another mutant defective in many or all tylosin biosynthetic reactions, but deleted for the AUD and other DNA sequences. These findings suggest that tylosin structural genes, several genes encoding antibiotic resistance determinants, and amplifiable DNA are present on a self-transmissible element that does not mobilize chromosomal genes, and that JS85 and JS87 contain deletions, and JS85 an amplification, of overlapping portions of this element.     

Influence of cyclic AMP on the growth response and anaerobic metabolism of carbon monoxide in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus strain 1 (str. 1), a photoheterotrophic bacterium, used CO as an energy substrate under anaerobic CO/light conditions, and exhibited a diauxic growth response when CO was removed from the culture. Changes in the level of cyclic AMP which occurred in cells during diauxie suggested that the cyclic nucleotide operated as an intracellular control molecule. During CO/light-phase growth, intracellular cyclic AMP was 30 pmol/mg protein, and, as str. 1 adapted for photosynthetic growth after removal of CO, intracellular cyclic AMP levels decreased to 9 pmol/mg protein. Reexposure of a light culture to CO induced synthesis of CO oxidation activity (measured as CO:MV oxidoreductase). If 10 mM cyclic AMP was added with CO, the rate of synthesis of CO:MV oxidoreductase activity increased 25-fold, and str. 1 produced 1,230 units of activity (nmol CO oxidized min^-1 mg^-1 protein) after only 1 h. With cyclic AMP and no CO, no incerease in CO oxidation activity was seen. Appearance of CO oxidation activity in str. 1 represented de novo protein synthesis and was blocked with chloramphenicol. In addition to stimulating formation of CO oxidative activity, a high level of cyclic AMP in str. 1 during growth with CO appeared to influence photometabolism negatively by repressing bacteriochlorophyll formation.Abbreviations Bchl a bacteriochlorophyll a - MV methyl viologen - CO MV oxidoreductase, carbon monoxide: methyl viologen oxidoreductase     

Inaccurate quantitation of palmitate in metabolomics and isotope tracer studies due to plastics

Introduction

Palmitate, the typical end product released from fatty acid synthase, is of interest to many researchers performing metabolomics. Although palmitate can be readily detected by using mass spectrometry, many metabolomic platforms involve the use of plastic consumables that introduce a competing background signal of palmitate.

Objectives

The goal of this study was to quantify palmitate contamination in metabolomics and isotope tracer studies and to examine the reliability of approaches for reducing error.

Methods

We measured the quantitative error introduced by palmitate contamination from 4 vendors of plastic consumables used in combination with several different extraction solvents.

Results

The background palmitate signal was as much as sixfold higher than the biological palmitate signal from 4 million 3T3-L1 cells. Importantly, the palmitate contamination signal was highly variable between plastic consumables (even within the same lot) and therefore could not be accurately removed by subtracting the background as measured from a blank. In addition to affecting relative and absolute quantitation, the palmitate background signal from disposable plastics also led to the underestimation of labeled palmitate in isotope tracer experiments.

Conclusion

When measuring palmitate standard solutions, the best results were obtained when glass vials and glass pipettes were used. However, much of the palmitate background signal could be eliminated by pre-rinsing plastic vials and plastic pipette tips with methanol prior to sample introduction. For isotope tracer studies, error could also be minimized by estimating palmitate enrichment from palmitoylcarnitine, which does not have a competing contamination signal from plastic consumables.

     

Sporadic Isolation of Sabin-Like Polioviruses and High-Level Detection of Non-Polio Enteroviruses during Sewage Surveillance in Seven Italian Cities,after Several Years of Inactivated Poliovirus Vaccination

Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5′ noncoding region (5′NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile > Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing.