Afamin is a novel human vitamin E-binding glycoprotein characterization and in vitro expression

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.     

Biochemical characterization of rat intestine development using high-resolution magic-angle-spinning 1H NMR spectroscopy and multivariate data analysis

We report details of metabolic profiles for small intestinal samples obtained using high-resolution magic-angle-spinning (HRMAS) (1)H NMR spectroscopy. Intact samples of jejunum and ileum from male Long Evans rats were analyzed on a 600 MHz spectrometer using standard one and two-dimensional (1)H NMR spectroscopic pulse sequences. The metabolic profiles of ileum and jejunum predominantly comprised a number of amino acids, lipids, glycerophosphocholine (GPC), choline, creatine, and ethanol, a number of carboxylic acids including acetate and lactate, and nucleoside bases including cytosine, isocytosine, and uracil. Principal component analysis (PCA) was applied to these NMR data to characterize the biochemical differences between jejunum and ileum tissues. Compared with ileum, jejunum contained higher levels of lipids, GPC, choline, lactate and creatinine, but lower levels of amino acids and acetate. In addition, the age dependence of the biochemical composition of intestinal tissues from young rats (15, 36 days and 3-4 months old) was studied. In general, levels of lipids, lactate, taurine and creatinine were positively correlated with age while amino acids and GPC decreased in the older age group. This study will provide a metabolic reference for further studies assessing the metabolic consequences of nutrition, stress and gut microbiota on intestinal composition.     

Detection of hypothetical proteins in human fetal perireticular nucleus

There is a legion of hypothetical proteins (HP) in prokaryotic and eukaryotic proteomes and the aim of this study was to describe HP in the perireticular nucleus (PN), a key structure in human brain development. Tissue from four PNs was homogenized and extracted proteins were run on two-dimensional gel electrophoresis followed by in-gel digestion and mass spectrometrical identification of proteins. Several databases were used for obtaining bioinformatic information and searching for functional and structural domains. Five spots represented HP: KIAA0423 protein (Q9Y4F4), hypothetical protein KIAA0153 (Q14166), hypothetical protein DKFZp564A2416 (Q9NTW4), hypothetical protein DKFZp564H1122 (Q9H0W9), and hypothetical protein DKFZp564D1378 (Q9H0R4). These structures were predicted to serve in cell cycle, DNA-condensation, neurogenesis, or apoptosis. The existence of formerly HP proteins in the PN of human fetal brain is shown, thus extending knowledge of the brain proteome and proposing the method used as a suitable analytical tool for searching HP.     

Cell division defects of Schizosaccharomyces pombe liz1- mutants are caused by defects in pantothenate uptake

The liz1⁺ gene of the fission yeast Schizosaccharomyces pombe was previously identified by complementation of a mutation that causes abnormal mitosis when ribonucleotide reductase is inhibited. Liz1 has similarity to transport proteins from Saccharomyces cerevisiae, but the potential substrate and its connection to the cell division cycle remain elusive. We report here that liz1⁺ encodes a plasma membrane-localized active transport protein for the vitamin pantothenate, the precursor of coenzyme A (CoA). Liz1 is required for pantothenate uptake at low extracellular concentrations. A lack of pantothenate uptake results in three phenotypes: (i) slow growth, (ii) delayed septation, and (iii) aberrant mitosis in the presence of hydroxyurea (HU). All three phenotypes are suppressed by high extracellular concentrations of pantothenate, where pantothenate uptake occurs by passive diffusion. liz1Δ mutants are viable because they can synthesize pantothenate from uracil as an endogenous source. The use of uracil for both pantothenate biosynthesis and deoxyribonucleotide generation provides an explanation for the aberrant mitosis in the presence of HU. HU blocks ribonucleotide reductase, and we propose that the accumulation of ribonucleotides reduces uracil biosynthesis by feedback inhibition of aspartate transcarbamoylase. Thus, the addition of HU to liz1Δ mutants results in a shortage of pantothenate. Because liz1Δ mutants show striking similarities to mutants with defects in fatty acid biosynthesis, we propose that the shortage of pantothenate compromises fatty acid synthesis, resulting in slow growth and mitotic defects.     

Self-assembly and dynamics of poly(gamma-benzyl-l-glutamate) peptides

The structure and the associated dynamics have been investigated in a series of oligopeptides of gamma-benzyl-l-glutamate using DSC, WAXS, FTIR, NMR and dielectric spectroscopy, and rheology, respectively. The peptides with degrees of polymerization below 18 are mixtures of a lamellar assembly of beta sheets and of columnar hexagonal arrangement of alpha helices, whereas for longer chains, the intramolecular hydrogen bonds stabilize only the alpha-helical conformations. Multiple dielectrically active processes were found. Starting from low temperatures, the two Arrhenius processes (gamma and beta), with apparent activation energies of 20.6 and 50.2 kJ/mol, respectively, associate with the local relaxation of the side-chain methylene units (gamma process) and with more cooperative motions of the side chain dipoles sensitive to the 7/2 helical packing (beta process). The glass transition is manifested in the thermal properties with a step in the heat capacity and with an intense dielectric process bearing characteristics (molecular weight dependence, temperature dependence of relaxation times) known from amorphous polymers. Based on these findings, the alpha process is attributed to the relaxation of amorphous segments located between and at the end of helically ordered segments. Two slower processes were identified with opposite molecular weight dependence. The weak intermediate mode with an M2 molecular weight dependence of the characteristic relaxation times suggests amorphous-like chains, whereas the strong slower process originates from the loss of dipole orientational capacity caused by structural defects and reflects the migration of helical sequences along the chains. This identifies the helices as structures extending over rather short fragments of chains (i.e., of low persistence length). The viscoelastic response indicated that the structural defects arise from locally aggregated chains that inhibit the flow of oligopeptides.     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.     

Measurement of exhaled hydrogen peroxide from rabbit lungs

Exhaled H2O2 is considered an indicator of lung inflammatory and oxidative stress. Moreover, H2O2 may be involved in signal transduction processes. It is not fully elucidated to what extent (i) H2O2 escapes from the intravascular compartment, and (ii) pulmonary H2O2 generation and nasopharyngeal H2O2 generation contribute to exhaled H2O2. We investigated H2O2 concentrations in breath condensate from isolated buffer-perfused and ventilated rabbit lungs, and from both intubated and spontaneously breathing rabbits with a horseradish peroxidase/2',7'dichlorofluorescin assay. For the perfused lungs, a H2O2 concentration of 58 +/- 19 nM was found. Addition of H2O2 to the buffer fluid resulted in only minute appearance in the exhaled air (<0.001%). Levels of exhaled H2O2 in intubated rabbits and perfused lungs were virtually identical. Nearly ten-fold higher levels were detected in spontaneously breathing rabbits. Decreasing the inspired oxygen concentration from 21% to 1% resulted in a tendency toward decreased H2O2 exhalation in perfused lungs. In contrast, phorbol-12-myristate-13-acetate (PMA) prompted a approximately 4-fold increase in H2O2 exhalation. We conclude that the horseradish peroxidase/2',7'dichlorofluorescin assay is a feasible technique to measure H2O2 in exhaled breath condensate in rabbits. When collecting exhaled air via the tracheal tube, the signal represents pulmonary H2O2 generation with the contribution of the remaining body being negligible.     

Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity

In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.