The puromycin route to assess stereo- and regiochemical constraints on peptide bond formation in eukaryotic ribosomes

We synthesized a series of puromycin analogues to probe the chemical specificity of the ribosome in an intact eukaryotic translation system. These studies reveal that both d-enantiomers and beta-amino acid analogues can be incorporated into protein, and provide a quantitative means to rank natural and unnatural residues. Modeling of a d-amino acid analogue into the 50S ribosomal subunit indicates that steric clash may provide part of the chiral discrimination. The data presented provide one metric of the chiral and regiospecificity of mammalian ribosomes.     

Determination of plutonium isotope ratios at very low levels by ICP-MS using on-line electrochemically modulated separations

Electrochemically modulated separations (EMS) are shown to be a rapid and selective means of extracting and concentrating Pu from complex solutions prior to isotopic analysis by inductively coupled plasma mass spectrometry (ICP‐MS). This separation is performed in a flow injection mode, on‐line with the ICP‐MS. A three‐electrode, flow‐by electrochemical cell is used to accumulate Pu at an anodized glassy carbon electrode by redox conversion of Pu(III) to Pu (IV&VI). The entire process takes place in 2% (v/v) (0.46 M) HNO₃. No redox chemicals or acid concentration changes are required. Plutonium accumulation and release is redox dependent and controlled by the applied cell potential. Large transient volumetric concentration enhancements can be achieved. Based on more negative U(IV) potentials relative to Pu(IV), separation of Pu from uranium is efficient, thereby eliminating uranium hydride interferences. EMS‐ICP‐MS isotope ratio measurement performance will be presented for femtogram to attogram level plutonium isotope injections.     

A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.     

Lymph Nodes Draining Infections Investigated by PET and Immunohistochemistry in a Juvenile Porcine Model

Background: [¹⁸F]FDG and [¹¹C]methionine accumulate in lymph nodes draining S. aureus -infected foci. The lymph nodes were characterized by weight, [¹¹C]methionine- and [¹⁸F]FDG-positron emissions tomography (PET)/computed tomography (CT), and immunohistochemical (IHC)-staining. Methods: 20 pigs inoculated with S. aureus into the right femoral artery were PET/CT-scanned with [¹⁸F]FDG, and nine of the pigs were additionally scanned with [¹¹C]methionine. Mammary, medial iliac, and popliteal lymph nodes from the left and right hind limbs were weighed. IHC-staining for calculations of area fractions of Ki-67, L1, and IL-8 positive cells was done in mammary and popliteal lymph nodes from the nine pigs. Results: The pigs developed one to six osteomyelitis foci. Some pigs developed contiguous infections of peri-osseous tissue and inoculation-site abscesses. Weights of mammary and medial iliac lymph nodes and their [¹⁸F]FDG maximum Standardized Uptake Values (SUV_FDGmax) showed a significant increase in the inoculated limb compared to the left limb. Popliteal lymph node weight and their FDG uptake did not differ significantly between hind limbs. Area fractions of Ki-67 and IL-8 in the right mammary lymph nodes and SUV_Metmax in the right popliteal lymph nodes were significantly increased compared with the left side. Conclusion: The PET-tracers [¹⁸F]FDG and [¹¹C]methionine, and the IHC- markers Ki-67 and IL-8, but not L1, showed increased values in lymph nodes draining soft tissues infected with S. aureus. The increase in [¹¹C]methionine may indicate a more acute lymph node response, whereas an increase in [¹⁸F]FDG may indicate a more chronic response.     

Self‐Assembly of Differently Shaped Protein–Polymer Conjugates through Modification of the Bioconjugation Site

Self‐assembly of protein–polymer block copolymers is an attractive route for preparing biocatalytic materials. To clarify the effect of bioconjugate shape on self‐assembly without changing the chemical details of either block, four different conjugation sites are selected on the surface of the model globular protein mCherry at residues 3, 108, 131, and 222 to alter the colloidal shape of the bioconjugate. All four mCherry‐b‐poly(N‐isopropylacrylamide) bioconjugates show qualitatively similar phase diagrams, indicating that self‐assembly is robust with respect to changes in conjugation site. However, protein orientation has an effect on the location of the order–disorder transition concentration, and the stability of the disordered micellar phase is shown to be different for each conjugate. Differences in domain spacing also suggest changes in protein orientation within the lamellae.

     

Thermal Isomerization of 7,8-Diazabicyclo[4.2.2]deca-2,4,7,9-tetraene N-oxide to Benzaldehyde Oxime

Thermal transformation of 7,8-diazabicyclo[4.2.2]deca-2,4,7,9-tetraene N-oxide ( 3 ) was observed, (Z)-benzaldehyde oxime being the major product, with (E)-benzaldehyde oxime as a minor product. N-Oxide 3 was labeled with deuterium α to the oxidized N-atom. The location of the deuterium label in the thermolysis products fitted one of two reasonable mechanisms.