Purification of a soluble, sodium-nitroprusside-stimulated guanylate cyclase from bovine lung

A soluble, sodium-nitroprusside-stimulated guanylate cyclase as been purified from bovine lung by DEAE-cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20, w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 mumol of cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 microM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 mumol min-1 mg-1.     

Hairy cell leukemia: kinetics and intercellular relationships of cells in leukemic colonies grown in a semisolid medium

We examined cellular relationships and cytokinetics of hairy cells in colonies formed in an in vitro cloning system. Cells in small colonies and at the periphery of larger ones were separated by wide, irregular intercellular spaces. Cells in the core of large colonies were elaborately intertwined by their cytoplasmic processes and so densely packed that their intercellularity was greatly reduced. Cell labeling with 3H-thymidine revealed indices ranging from less than 1% to 35%. The combination of autoradiography with a stain for tartrate-resistant acid phosphatase showed that the enzyme was not expressed by cells in S-phase. The cellular relationships of hairy cells in colonies grown in this system closely mimic those of their in vivo counterparts in the spleen and bone marrow.     

Subdivisions of the terminal nerve in Xenopus laevis

The majority of recent studies on the terminal nerve (nt) in various vertebrates either involved tracer injections into the nasal cavity or made use of the LHRH-/FMRFamide-like immunoreactivity (ir) of a portion of its fibers. The present investigation was designed to determine the extent of overlap between data rendered by the two methods in Xenopus. The findings reveal no overlap of nt projections visualized by the two experimental techniques. This result sheds doubt on the validity of current definitions of the nt system.     

‘You are inferior!’ Revisiting the expressivist argument

According to the expressivist argument the choice to use biotechnologies to prevent the birth of individuals with specific disabilities is an expression of disvalue for existing people with this disability. The argument has stirred a lively debate and has recently received renewed attention. This article starts with presenting the expressivist argument and its core elements. It then goes on to present and examine the counter‐arguments before it addresses some aspects that have gained surprisingly little attention. The analysis demonstrates that the expressivist argument has a wide range of underpinnings and that counter‐arguments tend to focus on only a few of these. It also reveals an important aspect that appears to have been ignored, i.e., that people do not select foetuses based on chromosomes or other biological traits, but based on characteristics of living persons with specific disabilities. This makes it more difficult to undermine the claim that negative selection of foetuses expresses a disvaluing of persons with such disabilities. It leaves the expressivist argument with a strong bite still.     

New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense

The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physicochemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression of foreign proteins via genetic fusion to magnetosome anchor peptides. However, the available genetic tool set for strong and controlled protein expression in magnetotactic bacteria is very limited. Here, we describe versatile vectors for either inducible or high-level constitutive expression of proteins in M. gryphiswaldense. The combination of an engineered native P_mamDC promoter with a codon-optimized egfp gene (Mag-egfp) resulted in an 8-fold increase in constitutive expression and in brighter fluorescence. We further demonstrate that the widely used P_tet promoter is functional and tunable in M. gryphiswaldense. Stable and uniform expression of the EGFP and β-glucuronidase (GusA) reporters was achieved by single-copy chromosomal insertion via Tn5-mediated transposition. In addition, gene duplication by Mag-EGFP–EGFP fusions to MamC resulted in further increased magnetosome expression and fluorescence. Between 80 and 210 (for single MamC–Mag-EGFP) and 200 and 520 (for MamC–Mag-EGFP–EGFP) GFP copies were estimated to be expressed per individual magnetosome particle.     

Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.