Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment.

BACKGROUND: Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide. METHODS: To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS). RESULTS: [(3)H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters. CONCLUSIONS: The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-κß and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis.

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration.

The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.

Molecular characterization, tissue-specific expression and RNAi knockdown of the first vitellogenin receptor from a tick.

This is the first full-length message for a vitellogenin receptor (VgR) sequenced from ticks. VgRs, members of the low-density lipoprotein receptor (LDLR) superfamily, mediate the uptake of the yolk protein, vitellogenin (Vg), from the hemolymph. The VgR message from the American dog tick, Dermacentor variabilis (GenBank accession No. DQ103506.4) comprised 5673 bp which coded for a 1798 aa deduced protein with a predicted 196.6 kDa molecular mass. After removing the 20 aa signal peptide, the 1778 aa deduced mature protein had a predicted 196.6 kDa molecular mass. BLAST comparisons showed the highest similarity to the VgR of the cockroach, Periplaneta americana. VgR message was expressed in mated female ovary but absent in female midgut and salivary glands or whole body mRNA from blood fed males, indicating that it is both sex and tissue specific. VgR transcript was absent in virgin (previtellogenic) females but present in ovaries of mated females following drop off. RNAi showed that unfed adult ticks injected with a VgR-dsRNA probe failed to lay eggs, develop brown eggs or fully express VgR transcript (Northern blots). In contrast, controls oviposited numerous normal brown eggs and showed strong expression of VgR transcripts. These results show that the expression of the VgR message is essential for Vg uptake and egg development in the American dog tick.

The flounder organic anion transporter fOat has sequence, function, and substrate specificity similarity to both mammalian Oat1 and Oat3.

The flounder renal organic anion transporter (fOat) has substantial sequence homology to mammalian basolateral organic anion transporter orthologs (OAT1/Oat1 and OAT3/Oat3), suggesting that fOat may have functional properties of both mammalian forms. We therefore compared uptake of various substrates by rat Oat1 and Oat3 and human OAT1 and OAT3 with the fOat clone expressed in Xenopus oocytes. These data confirm that estrone sulfate is an excellent substrate for mammalian OAT3/Oat3 transporters but not for OAT1/Oat1 transporters. In contrast, 2,4-dichlorophenoxyacetic acid and adefovir are better transported by mammalian OAT1/Oat1 than by the OAT3/Oat3 clones. All three substrates were well transported by fOat-expressing Xenopus oocytes. fOat K(m) values were comparable to those obtained for mammalian OAT/Oat1/3 clones. We also characterized the ability of these substrates to inhibit uptake of the fluorescent substrate fluorescein in intact teleost proximal tubules isolated from the winter flounder (Pseudopleuronectes americanus) and killifish (Fundulus heteroclitus). The rank order of the IC(50) values for inhibition of cellular fluorescein accumulation was similar to that for the K(m) values obtained in fOat-expressing oocytes, suggesting that fOat may be the primary teleost renal basolateral Oat. Assessment of the zebrafish (Danio rerio) genome indicated the presence of a single Oat (zfOat) with similarity to both mammalian OAT1/Oat1 and OAT3/Oat3. The puffer fish (Takifugu rubripes) also has an Oat (pfOat) similar to mammalian OAT1/Oat1 and OAT3/Oat3 members. Furthermore, phylogenetic analyses argue that the teleost Oat1/3-like genes diverged from a common ancestral gene in advance of the divergence of the mammalian OAT1/Oat1, OAT3/Oat3, and, possibly, Oat6 genes.

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say).

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.