Characterization of the ugp region containing the genes for the phoB dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary The ugp structural genes, coding for the pho regulon dependent sn-glycerol-3-phosphate transport system, were cloned in pBR322 and characterized. The expression of the cloned ugp system was phoB dependent. Cells containing the ugp plasmid overproduced the G3P binding protein upon phosphate starvation. Tn5 mutagenesis of the cloned DNA revealed that the ugp genes are organized in two separate operons which comprise at least four genes: ugpB and ugpD constitute one operon, ugpA and ugpC constitute the other. The structural gene for the G3P binding protein (G3PBP) is ugpB.The ugpC gene product was also synthesized in minicells as a polypeptide, with an apparent molecular weight of 40,000. No gene products could be assigned to the ugpA and ugpD genes. Hybridization experiments allowed the physical characterization of 20 kb of DNA adjacent to the ugp genes on the E. coli chromosome including the liv genes.     

Immunocytochemical localization of two peroxisomal enzymes of lipid beta-oxidation in specific granules of rat eosinophils

Peroxisomes contain a system for beta-oxidation of fatty acids which differs from the mitochondrial system and is associated with hydrogen peroxide formation. We show that two enzymes: enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase of the peroxisomal system are present in specific granules of rat eosinophils. Both enzyme proteins were purified from rat liver and monospecific antibodies were raised in rabbits. Eosinophils from peripheral blood and tissue eosinophils from the wall of intestine, fixed by glutaraldehyde and embedded in Epon were investigated. The postembedding immunocytochemical procedure with protein A-gold technique was used. The gold particles representing the antigenic sites for both enzymes were present only in specific granules of eosinophils with no immune deposits in mitochondria, nucleus and the cytoplasm. Although gold particles were found over the entire domain of the granule, the electron dense paracrystalline inclusions contained more gold than the granule matrix. Control preparations incubated with nonspecific IgG and protein A-gold complex alone were negative. These findings indicate that in specific granules of eosinophils both peroxisomal and lysosomal enzymes share the same intracellular compartment. The peroxisomal lipid beta-oxidation in eosinophils may be involved in generation of hydrogen peroxide, which has a crucial role in killing of metazoon parasites.     

Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn²⁺ ion as the active-site centre whereas others are only active with Fe²⁺ (or Co²⁺, Ni²⁺) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.     

CD8+ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2

A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8⁺ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8⁺ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8⁺ T lymphocytes.     

Review of pharmacological therapies in fibromyalgia syndrome

This review addresses the current status of drug therapy for the management of fibromyalgia syndrome (FMS) and is based on interdisciplinary FMS management guidelines, meta-analyses of drug trial data, and observational studies. In the absence of a single gold-standard medication, patients are treated with a variety of drugs from different categories, often with limited evidence. Drug therapy is not mandatory for the management of FMS. Pregabalin, duloxetine, milnacipran, and amitriptyline are the current first-line prescribed agents but have had a mostly modest effect. With only a minority of patients expected to experience substantial benefit, most will discontinue therapy because of either a lack of efficacy or tolerability problems. Many drug treatments have undergone limited study and have had negative results. It is unlikely that these failed pilot trials will undergo future study. However, medications, though imperfect, will continue to be a component of treatment strategy for these patients. Both the potential for medication therapy to relieve symptoms and the potential to cause harm should be carefully considered in their administration.The desire to take medicine is perhaps the greatest feature which distinguishes man from animals.Sir William Osler (1849–1919)     

Differential activity of innate defense antimicrobial peptides against <Emphasis Type=Italic>Nocardia</Emphasis> species

Background  

Members of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human α-defensins human neutrophil peptides (HNPs) 1-3, human β-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine β-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated.     

Proteolytic enzymes in Chlamydomonas I. A survey on the aminopeptidase pattern in asynchronous vegetative cells of Chlamydomonas reinhardii

The supernatant of a crude extract from vegetative cells ofChlamydomonas reinhardii contains three different types of aminopeptidases.They are similar in their substrate specificities to the relativealanine specific aminopeptidases, the relative leucine specificaminopeptidases and the specific proline iminopeptidases describedin many other systems. Relative alanine specific aminopeptidasewhich also cleaves N-terminal Lys and Leu residues has a molecularweight of 92,000 daltons and is inhibited by zinc and manganeseions.Relative leucine specific aminopeptidase shows high activitywith N-terminal Phe besides Leu, and is capable of cleavingTyr, Pro, and to a minor degree Ala. It has a molecular weightof 76,000 daltons. No effects on its activity were detectedin the presence of divalent cations or chelating agents. Theiminopeptidase specifically splits N-terminal Pro and has amolecular weight of about 255,000 daltons. All the enzymes showoptimal activity at pH 8.0–8.5. The two aminopeptidases can be separated from the iminopeptidaseby ammonium sulfate solubilization and from each other by subsequentfractionation on DEAE-cellulose. Relative leucine specific activityappeared as a single enzyme in all the fractionation techniquesused, but it gave two distinct bands when crude extracts wererun on native polyacrylamide gels. Therefore, this enzyme mayexist in multiple molecularforms. (Received October 17, 1978; )     

A dendrite-autonomous mechanism for direction selectivity in retinal starburst amacrine cells

Detection of image motion direction begins in the retina, with starburst amacrine cells (SACs) playing a major role. SACs generate larger dendritic Ca²⁺ signals when motion is from their somata towards their dendritic tips than for motion in the opposite direction. To study the mechanisms underlying the computation of direction selectivity (DS) in SAC dendrites, electrical responses to expanding and contracting circular wave visual stimuli were measured via somatic whole-cell recordings and quantified using Fourier analysis. Fundamental and, especially, harmonic frequency components were larger for expanding stimuli. This DS persists in the presence of GABA and glycine receptor antagonists, suggesting that inhibitory network interactions are not essential. The presence of harmonics indicates nonlinearity, which, as the relationship between harmonic amplitudes and holding potential indicates, is likely due to the activation of voltage-gated channels. [Ca²⁺] changes in SAC dendrites evoked by voltage steps and monitored by two-photon microscopy suggest that the distal dendrite is tonically depolarized relative to the soma, due in part to resting currents mediated by tonic glutamatergic synaptic input, and that high-voltage–activated Ca²⁺ channels are active at rest. Supported by compartmental modeling, we conclude that dendritic DS in SACs can be computed by the dendrites themselves, relying on voltage-gated channels and a dendritic voltage gradient, which provides the spatial asymmetry necessary for direction discrimination.