Optimal induction of T-cell responses against hepatitis C virus E2 by antigen engineering in DNA immunization

Although DNA immunization is a safe and efficient method for inducing cellular immune responses, it generates relatively weak and slow immune responses. Here, we investigated the effect of hepatitis C virus (HCV) antigen modifications on the induction of T-cell responses in DNA immunization. It is likely that the strength of T-cell responses has an inverse relationship with the length of the insert DNA. Interestingly, a mixture of several plasmids carrying each gene induced a higher level of T-cell responses than a single plasmid expressing a long polyprotein. Moreover, the presence of a transmembrane domain in HCV E2 resulted in stronger T-cell responses against E2 protein than its absence. Taken together, our results indicate that the tailored modifications of DNA-encoded antigens are capable of optimizing the induction of T-cell responses which is required for eliminating the cells chronically infected with highly variable viruses such as HCV and human immunodeficiency virus.     

Follicular dendritic cell dedifferentiation by treatment with an inhibitor of the lymphotoxin pathway dramatically reduces scrapie susceptibility

Transmissible spongiform encephalopathies (TSEs) may be acquired peripherally, in which case infectivity usually accumulates in lymphoid tissues before dissemination to the nervous system. Studies of mouse scrapie models have shown that mature follicular dendritic cells (FDCs), expressing the host prion protein (PrP(c)), are critical for replication of infection in lymphoid tissues and subsequent neuroinvasion. Since FDCs require lymphotoxin signals from B lymphocytes to maintain their differentiated state, blockade of this stimulation with a lymphotoxin beta receptor-immunoglobulin fusion protein (LT beta R-Ig) leads to their temporary dedifferentiation. Here, a single treatment with LT beta R-Ig before intraperitoneal scrapie inoculation blocked the early accumulation of infectivity and disease-specific PrP (PrP(Sc)) within the spleen and substantially reduced disease susceptibility. These effects coincided with an absence of FDCs in the spleen for ca. 28 days after treatment. Although the period of FDC dedifferentiation was extended to at least 49 days by consecutive LT beta R-Ig treatments, this had little added protective benefit after injection with a moderate dose of scrapie. We also demonstrate that mature FDCs are critical for the transmission of scrapie from the gastrointestinal tract. Treatment with LT beta R-Ig before oral scrapie inoculation blocked PrP(Sc) accumulation in Peyer's patches and mesenteric lymph nodes and prevented neuroinvasion. However, treatment 14 days after oral inoculation did not affect survival time or susceptibility, suggesting that infectivity may have already spread to the peripheral nervous system. Although manipulation of FDCs may offer a potential approach for early intervention in peripherally acquired TSEs, these data suggest that the duration of the treatment window may vary widely depending on the route of exposure.     

A case of acute gastric anisakiasis provoking severe clinical problems by multiple infection

Acute gastric anisakiasis with multiple anisakid larvae infection is reported. A 68-year-old woman residing in Busan, Korea, had epigastric pain with severe vomiting about 5 hours after eating raw anchovies. Four nematode larvae penetrating the gastric mucosae in the great curvature of the middle body and fundus were found and removed during gastro-endoscopic examination. Another one thread-like moving larva was found in the great curvature of upper body on the following day. On the basis of their morphology, the worms were identified as the 3rd stage larvae of Anisakis simplex. This case is acute gastric anisakiasis provoking severe clinical problems by the multiple infection and the greatest number of anisakid larvae found in a patient in Korea.     

Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions

The secreted protein toxin produced by Bacillus anthracis contributes to virulence of this pathogen and can cause many of the symptoms seen during an anthrax infection, including shock and sudden death. The cell-binding component of anthrax toxin, protective antigen, mediates entry of the toxin into cells by first binding directly to the extracellular integrin-like inserted (I) domain of the cellular anthrax toxin receptor, ATR. Here we report that this interaction requires an intact metal ion-dependent adhesion site (MIDAS) in the receptor as well as the presence of specific divalent cations. Also, we demonstrate that the toxin-receptor interaction is critically dependent on the Asp-683 carboxylate group of protective antigen, which projects from the receptor binding surface. We propose that this carboxylate group completes the coordination of the MIDAS metal of ATR, mimicking integrin-ligand interactions.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.     

Protein kinase C nu/protein kinase D3 nuclear localization,catalytic activation,and intracellular redistribution in response to G protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC micro/PKD, PKD2, and PKCnu/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.