Mechanisms of internalization and recycling of the chemokine receptor, CCR5.

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C(3) exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.     

Characterisation of Recombinant Serotonin 5-HT1A Receptors Expressed in Chinese Hamster Ovary Cells: The Agonist [3H]Lisuride Labels Free Receptor and Receptor Coupled to G Protein

Abstract: Cholinesterases form a family of serine esterases that arise in animals from at least two distinct genes. Multiple forms of these enzymes can be precisely localized and regulated by alternative mRNA splicing and by co- or posttranslational modifications. The high catalytic efficiency of the cholinesterases is quelled by certain very selective reversible and irreversible inhibitors. Owing largely to the important role of acetylcholine hydrolysis in neurotransmission, cholinesterase and its inhibitors have been studied extensively in vivo. In parallel, there has emerged an equally impressive enzyme chemistry literature. Cholinesterase inhibitors are used widely as pesticides; in this regard the compounds are beneficial with concomitant health risk. Poisoning by such compounds can result in an acute but usually manageable medical crisis and may damage the CNS and the PNS, as well as cardiac and skeletal muscle tissue. Some inhibitors have been useful for the treatment of glaucoma and myasthenia gravis, and others are in clinical trials as therapy for Alzheimer's dementia. Concurrently, the most potent inhibitors have been developed as highly toxic chemical warfare agents. We review treatments and sequelae of exposure to selected anticholinesterases, especially organophosphorus compounds and carbamates, as they relate to recent progress in enzyme chemistry.     

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub‐populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi‐potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell‐enriched population showed the most pronounced deregulation of migration‐associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.     

Lipid‐associated aggregate formation of superoxide dismutase‐1 is initiated by membrane‐targeting loops

Copper‐Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo‐SOD1 aggregates may be due to their ability to cause defects within cell membranes by co‐aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane‐bound copper chaperones. But how apo‐SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc‐binding loops in apo‐SOD1 dimers play a critical role in the binding of 1‐octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo‐SOD1 monomer also associates with phospholipid bilayer via its zinc‐binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane‐bound copper chaperone. The orientation also suggests how membrane‐bound monomers could act as seeds for membrane‐associated SOD1 aggregation. Proteins 2014; 82:3194–3209. © 2014 Wiley Periodicals, Inc.     

Antifungal activity of rhizospheric bacteria

Fluorescent Pseudomonad spp. were isolated from the rhizosphere of potato plants (Algeria) by serial dilutions of rhizosphere soils on Kings B medium and were tested for their antifungal activity. The antifungal activity of the Pseudomonas isolated from Potatoes rhizosphere was tested against Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum in dual culture with bacteria on PDA. The Petri dish was divided into tow, on one the bacteria was spread and on the opposite side fungal plugs were inoculated and incubated for one week. Fourteen bacteria were isolated; only one isolate inhibited the growth of Pythium ultimum, Rhizoctonia solani, Fusarium solani; Fusarium oxysporum f.sp. albedinis and Fusarium oxysporum f. sp. Lycopersici with inhibition zones of 39.9, 33.7, 30.8, 19.9 and 22.5 mm respectively.     

Production of Ascochyta rabiei lacking solanapyrone A toxin production

Ascochyta rabiei, agent of Ascochyta blight of chickpea produces three toxins, Solanapyrones A, B and C of which solanapyrone A is the most toxic. All isolates of the fungus so far examined produce at least one of the Solanapyrone toxins, usually Solanapyrone A. The universality of solanapyrone production argues strongly for the importance of the toxins in virulence or pathogenicity. However, further evidence for this awaits the development of mutants lacking toxin production. Generation and isolation of fungal mutants defective in pathogenicity has been very useful for understanding the genetic and enzymatic processes responsible for infectivity in a number of pathosystems. Numerous tools have been used to transform plants and micro-organisms but the most widely micro-organism employed is Agrobacterium tumefaciens. In the present experiments, two strains of A. tumefaciens, AGL1 and LBA1126, harbouring two different plasmids, both encoding a gene for hygromycin resistance in the T-DNA region were used to transform isolate Tk21 of A. rabiei. The transformation of Ascochyta rabiei, gave rise to 498 colonies which grew on media supplemented with the selective agent; hygromycin B. The 30 sporulated transformants produced solanapyrone A on the specific medium at different rates. Solanapyrone A production, as demonstrated by the absorption of light at 327 nm, varied from 2.11 microg/ml to 4.32 microg/ml, representing a reduction of 74.11% to 46.99% in comparison with the wild type (8.15 microg/ml).     

Mechanisms of haplotype divergence at the <Emphasis Type="Italic">RGA08</Emphasis> nucleotide-binding leucine-rich repeat gene locus in wild banana <Emphasis Type="Italic">(Musa balbisiana)</Emphasis>

Background  

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).     

Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.     

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.