Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Trypsin and α-amylase inhibitors are differentially induced in leaves of amaranth (Amaranthus hypochondriacus) in response to biotic and abiotic stress

Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an α -amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and α -amylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect α -amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in α -amylase inhibitor activity and AAI levels were detected in leaves of insect-damaged, MeJA- and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water- or salt-stress. A differential induction of trypsin inhibitor activity and α -amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and α -amylase inhibitors could protect A. hypochondriacus against multiple types of stress.     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use

We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.     

Some Characteristics of Threonine Transport Across the Blood-Brain Barrier of the Rat

Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system.     

Stopped-flow mechanistic study of bromide substitution by an organonitrile at an iron(II) phosphinic centre; a π-electron driven process

The kinetics of the displacement reactions of the bromide ligands of trans-[FeBr₂(depe)₂] (depe = Et₂PCH₂CH₂PEt₂) by the organonitrile NCCH₂C₆H₄OMe-4, in tetrahydrofuran (either in the absence or in the presence of added Br⁻), to give the corresponding mono- and dinitrile complexes trans-[FeBr(NCCH₂C₆H₄OMe-4)(depe)₂]⁺ and trans-[Fe(NCCH₂C₆H₄OMe-4)₂(depe)₂]²⁺, have been investigated by stopped-flow spectrophotometry. The substitution reaction occurs by a mechanism involving rate-limiting dissociation of bromo ligands to form the unsaturated intermediates [FeBr(depe)₂]⁺ (k₁ = 1.52 ± 0.02 s⁻¹) and [Fe(NCR)(depe)₂]²⁺ (k₃ = 0.063 ± 0.008 s⁻¹) which add the nitrile ligand to form those nitrile complexes. The competition between the nitrile and Br⁻ for such metal centres has also been investigated and a stronger inhibiting effect of added Br⁻ is observed for the substitution of the second bromo ligand relative to the first one. The kinetic data are rationalized in terms of π-electronic effects of these unsaturated metal centres and of the bromide and nitrile ligands.     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.