B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.     

Identification of tissue-specific microRNAs from mouse

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.     

The biophysics of leaf growth in salt-stressed barley. A study at the cell level

Biophysical parameters potentially involved in growth regulation were studied at the single-cell level in the third leaf of barley (Hordeum vulgare) after exposure to various degrees of NaCl stress for 3 to 5 d. Gradients of elongation growth were measured, and turgor pressure, osmolality, and water potentials (psi) were determined (pressure probe and picoliter osmometry) in epidermal cells of the elongation zone and the mature blade. Cells in the elongation zone adjusted to decreasing external psi through increases in cell osmolality that were accomplished by increased solute loads and reduced water contents. Cell turgor changed only slightly. In contrast, decreases in turgor also contributed significantly to psi adjustment in the mature blade. Solute deposition rates in the elongation zone increased at moderate stress levels as compared with control conditions, but decreased again at more severe NaCl exposure. Growth-associated psi gradients between expanding epidermal cells and the xylem were significant under control and moderate stress conditions (75 mM NaCl) but seemed negligible at severe stress (120 mM NaCl). We conclude that leaf cell elongation in NaCl-treated barley is probably limited by the rate at which solutes can be taken up to generate turgor, particularly at high NaCl levels.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.     

Zinc serum level in human immunodeficiency virus-infected patients in relation to immunological status

In human immunodeficiency virus (HIV) infection, serum level of zinc, an important micronutrient for immune function, is frequently diminished. The aim of this study was to determine the zinc status in relation to immunological parameters and disease stage in 79 HIV-1 seropositive patients. The median serum level of zinc was within normal limits (12.5 micromol/L) but in 23% of patients, zinc deficiency was seen. Decreased serum zinc was associated with a low CD4 cell count, high viral load, and increased neopterin and IgA levels. According to current treatment recommendations, the majority of patients received antiretroviral triple therapy. Zinc levels in treated and untreated patients were comparable. Referring to disease stage (CDC classification, 1993), the mean zinc level was highest in stage C and lowest in stage A. In conclusion, even under antiretroviral triple therapy, zinc deficiency is still of great importance in HIV infection, and zinc substitution in zinc deficient individuals should be taken into account to optimize therapeutical success.