Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (T_RM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8⁺ T_RM cells correlates with a better outcome. However, the requirements for generating and maintaining lung T_RM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the T_RM cell pool during RSV infection. We show that CD8⁺ T_RM cells expand independently from systemic CD8⁺ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8⁺ T_RM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored T_RM cell expansion upon re-challenge but failed to recover T_RM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8⁺ T_RM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.     

Brassica rapa plants adapted to microgravity with reduced photosystem I and its photochemical activity

The photosynthetic apparatus contains several protein complexes, many of which are regulated by environmental conditions. In this study, the influences of microgravity on PSI and PSII in Brassica rapa plants grown aboard the space shuttle were examined. We found that Brassica plants grown in space had a normal level of growth relative to controls under similar conditions on Earth. Upon return to Earth, cotyledons were harvested and thylakoid membranes were isolated. Analysis of chlorophyll contents showed that the Chl a/b ratio (3.5) in flight cotyledons was much higher than a ratio of 2.42 in the ground controls. The flight samples also had a reduction of PSI complexes and a corresponding 30% decrease of PSI photochemical activity. Immunoblotting showed that the reaction centre polypeptides of PSI were more apparently decreased (e.g. by 24-33% for PsaA and PsaB, and 57% for PsaC) than the light-harvesting complexes. In comparison, the accumulation of PSII complex was less affected in microgravity, thus only a slight reduction in D1, D2 and LHCII was observed in protein blots. However, there was a 32% decrease of OEC1 in the flight samples, indicating a defective OEC subcomplex. In addition, an average 54% increase of the 54 kDa CF1-beta isoform was found in the flight samples, suggesting that space-grown plants suffered from certain stresses, consistent with implications of the increased Chl a/b ratio. Taken together, the results demonstrated that Brassica plants can adapt to spaceflight microgravity, but with significant alterations in chloroplast structures and photosynthetic complexes, and especially reduction of PSI and its activity.     

Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.     

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.