The primary structure of Escherichia coli K12 2-deoxyribose 5-phosphate aldolase. Nucleotide sequence of the deoC gene and the amino acid sequence of the enzyme

The sequence of the deoC gene of Escherichia coli K12 and the amino acid sequence of the corresponding protein, deoxyriboaldolase, has been established. The protein consists of 259 amino acids with a molecular weight of 27 737. The purified enzyme may exist both as a monomer and as a dimer. On the basis of amino acid composition, molecular weight and catalytic properties, the enzymes from E. coli and Salmonella typhimurium seem to be almost similar. They belong to the class I aldolases, which form Schiff base intermediates. Using data for the S. typhimurium enzyme, the lysine residue involved in the active site in the E. coli enzyme was tentatively identified.     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Closed-loop system in evaluation of insulin infusion rates for an open-loop system.

The daily insulin requirements as calculated with a closed-loop system (Biostator), were used for insulin infusion with a portable, miniaturized pump in 6 juvenile-onset, insulin-requiring diabetics. Three diabetics were given insulin in a manner like that of the Biostator, e.g., maximal insulin infusion about 1 hour after start of meals. The MBG was 8.7 +/- 3.9 (SD) mmol/l. Three other diabetics had insulin in a fixed profile with peaks beginning simultaneously with meals. MBG of these patients was 4.4 +/- 1.9 mmol/l. Knowing the daily insulin dosage as calculated from the Biostator, normal blood glucose levels can be achieved with a fixed profile of insulin given by a portable pump.     

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.     

EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell–cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.     

Human palatal saliva: Adsorption behaviour and the role of low-molecular weight proteins

In situ ellipsometry was employed to study adsorption from human palatal saliva (HPalS) in terms of dependence on surface wettability and saliva concentration ( ? 1%). Adsorbed amounts, kinetics, and elutability with buffer and sodium dodecyl sulphate (SDS) were determined. The low-molecular weight protein content of bulk HPalS was also investigated using two-dimensional gel electrophoresis, and this revealed the presence of a large group of proteins < 100 kDa in size. Adsorption to pure (hydrophilic) and methylated (hydrophobized) silica surfaces revealed that the total adsorbed amounts were greater on hydrophobized silica. Below concentrations of 0.5 and 0.25% saliva, adsorption was concentration dependent on hydrophobized and hydrophilic surfaces, respectively. The initial adsorption ( ? 30 min) was faster on hydrophobized surfaces. Addition of SDS removed more material than buffer rinsing on both surfaces. Analysis of the adsorption kinetics indicated that the presence of low-molecular weight proteins plays a role in adsorption from HPalS.     

Life-and-Death Decisions in a Neonatal Intensive Care Unit in Denmark: The Discrete Authority of Origin Stories

In what ways are care and compassion implicated in efforts to establish lives worth living? Drawing on fieldwork in a Danish Neonatal Intensive Care Unit (NICU), in this article we investigate the role of family biographies in conducting life-and-death decisions around premature infants. Guided by a larger literature on citizenship, we view decisions in the NICU as political acts of assigning citizenship. We ask what bodies and biographies can generate and evoke care and compassion among NICU staff and forge entries or exits from the Danish Welfare State. We demonstrate that infants’ origin stories are appointed legitimate forms of suffering in contemporary Danish society and are thus granted an unnoticed form of authority in life-and-death decisions. In this way, we conclude that what comes to constitute a life worth living in the twenty-first-century Danish Welfare State is in fact the worth of the family.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.