Membrane Cholesterol Modulates the Outward Facing Conformation of the Dopamine Transporter and Alters Cocaine Binding

Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-β-cyclodextrin), we observed an increase in DAT binding B_max values for cocaine analogs [³H]WIN35428 and [¹²⁵I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function.     

Preexisting vaccinia virus immunity decreases SIV-specific cellular immunity but does not diminish humoral immunity and efficacy of a DNA/MVA vaccine

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.     

Epidemiological and clinical studies of ovine caseous lymphadenitis

A field survey was undertaken, to determine epidemiological, clinical and biological data on Ovine Caseous Lymphadenitis disease in 54 flocks originated from Sfax area in Tunisia. The mean morbidity rate of the cutaneous form was 5.1%. This form affected sheep over 3 months and under 2 years old. On the other hand, the mean morbidity rate of the visceral form, encountered in abattoirs, was 11.02%. The clinical aspect of the superficial (or cutaneous) form was often corresponding to one abscess, located particularly in the lymphatic nodes of the animal's head; while visceral (or internal) form of the disease was represented by the presence of a unique abscess found in the pulmonary lymphatic nodes. The macroscopic aspect of lesions showed that the size of abscess was comprised between 4 and 10 cm in diameter. At the cut, colour of the pus was white yellowish to yellow greyish with a fluid or a thick aspect like onion peels; pus was microscopic. Lesions were characterised by a fibrous shell, a pyogenic membrane and a necrotic center. Bacteriological research revealed that Corynebacterium pseudotuberculosis was the pathogen the most frequently isolated, followed by Staphylococcus aureus subsp anaerobius which was particularly found in sheep aged between 3 months and 2 years old.     

Mini-review highlighting toxic effects of triflumuron in insects and mammalian systems

Pesticides have been used to kill pests such as insects, fungi, rodents, and unwanted plants. As these compounds are potentially toxic to the target organisms, they could also be harmful to human health and the environment. Several chronic adverse effects have been identified even after months or years of exposure. The adverse effects of pesticides on the agricultural ecosystem have been a matter of concern in recent decades. In this review, we present an overview of the studies, including our previous studies, monitoring currently used pesticides in the Tunisian agricultural soils that belong to the class of insect growth regulators (IGRs). Triflumuron (TFM) is a benzoyl phenyl urea insecticide belonging to the class of IGRs. TFM is widely used around the world to increase crop yield by protecting them from damage caused by insects. TFM works by inhibiting the synthesis of chitin, an essential part of the insect cuticle, making it susceptible to pathogens and deformities. Consequently, insects become more susceptible to pathogens and malformations. However, studies revealing its toxicity and its mode of action in mammalian systems remain very limited. The aim of this review is to better inform the community about the impact of TFM on crops, the environment, and human beings by summarizing its toxic effects.     

Seasonal distribution and duration of the planktonic stage of Dover sole, Solea solea, larvae in the Bay of Biscay: an hypothesis

The possibility of deriving a prediction about the effect of seasonal variations in the duration of the planktonic larval phase on the dispersal of larval Dover sole was investigated. During six cruises, from February to May 1992, the distribution of sole larvae was studied along a 100-km transect, from the offshore spawning grounds to the coastal nurseries of the Bay of Biscay (France). Samples ( n = 189) were collected with a suprabenthic sampler, and vertical profiles of water temperature and salinity were recorded simultaneously. Counts of otolith increments of larval stage 4b (onset of metamorphosis) were used to estimate the duration of the planktonic life. Duration of the larval phase decreases by about 15 days (37%) with water temperature increase (between 8° in February and 11.2° C in May). Sole larvae occur from the coastal area to 100 km offshore. Within the same cruise, no difference in the duration of the planktonic life was observed between the larvae caught in the onshore and the offshore area. In spite of seasonal differences in abundance, the extent and the shape of the larval distributions during the period of study suggest that the seasonal variations in the duration of the planktonic life did not affect the larval distribution.     

Defining a novel ribonucleotide reductase r1 mRNA cis element that binds to an unique cytoplasmic trans-acting protein.

Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.     

Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.     

Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases

Apoptotic bodies can be used to target delivery of DNA-expressed immunogens into professional antigen-presenting cells (APCs). Here we show that antigen-laden apoptotic bodies created by vectors co-expressing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes and mutant caspase genes markedly increased T-cell responses. Both CD8 and CD4 T-cell responses were affected. The adjuvant activity was restricted to partially inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Active-site mutants of murine caspase 2 and an autocatalytic chimera of murine caspase 2 prodomain and human caspase 3 induced apoptosis that did not interfere with immunogen expression. The adjuvant activity also enhanced B-cell responses, but to a lesser extent than T-cell responses. The large increases in T-cell responses represent one of the strongest effects to date of a DNA adjuvant on cellular immunity.