Faecal parasites increase with age but not reproductive effort in wild female chimpanzees.

Energy investment in reproduction is predicted to trade off against other necessary physiological functions like immunity, but it is unclear to what extent this impacts fitness in long-lived species. Among mammals, female primates, and especially apes, exhibit extensive periods of investment in each offspring. During this time, energy diverted to gestation and lactation is hypothesized to incur short and long-term deficits in maternal immunity and lead to accelerated ageing. We examined the relationship between reproduction and immunity, as measured by faecal parasite counts, in wild female chimpanzees (Pan troglodytes schweinfurthii) of Kibale National Park, Uganda. While we observed higher parasite shedding (counts of eggs, cysts and larvae) in pregnant chimpanzees relative to cycling females, parasites rapidly decreased during early lactation, the most energetically taxing phase of the reproductive cycle. Additionally, while our results indicate that parasite shedding increases with age, females with higher fertility for their age had lower faecal parasite counts. Such findings support the hypothesis that the relatively conservative rate of female reproduction in chimpanzees may be protective against the negative effects of reproductive effort on health. This article is part of the theme issue 'Evolution of the primate ageing process'.

The WASP/Las17p-interacting protein Bzz1p functions with Myo5p in an early stage of endocytosis.

The formation of actin filaments is crucial for endocytosis and other interrelated cellular phenomena such as motility, polarized morphogenesis, and cytokinesis. In this paper we have investigated the role of the WASP/Las17-interacting protein Bzz1p in endocytosis and trafficking to the vacuole. We and others have recently shown that Bzz1p is an actin patch protein that interacts directly with Las17p via a SH3-polyproline interaction. Bzz1p functions with type I myosins to restore polarity of the actin cytoskeleton after NaCl stress. In an in vitro bead assay, GST-Bzz1p fusion protein triggers a functional actin polymerization machinery through its two C-terminal SH3 domains. In this paper we implicate Bzz1p with the type I myosins both in fluid-phase and in the internalization step of receptor-mediated endocytosis. As deduced from their localization as GFP fusions, the vacuolar delivery of endocytic and biosynthetic cargoes as well as the multivesicular body pathway appear unaffected. We further elucidate Bzz1p direct participation in actin polymerization by demonstrating that each of the SH3 domains of Bzz1p individually is able to trigger actin polymerization in a cell-free system dependent on Arp2/3, Las17p, Vrp1p, and the type I myosins. Taken together, our results show that Bzz1p participates, essentially via its SH3 domains, in early steps of endocytosis together with known actin nucleation activators.

SH3 domain-containing proteins and the actin cytoskeleton in yeast.

SH3 (Src homology-3) domains are involved in protein-protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)-SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott-Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast.

In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. We show that overexpression of either one of the two kinases Pkh1p or Pkh2p, that are homologous to mammalian 3-phosphoinositide-dependent kinase-1 (PDK1), can specifically suppress the sphingoid base synthesis requirement for endocytosis. Pkh1p and Pkh2p have an overlapping function because only a mutant with impaired function of both kinases is defective for endocytosis. Pkh1/2p kinases are activated in vitro by nanomolar concentrations of sphingoid base. These results suggest that Pkh1/2p kinases are part of a sphingoid base-mediated signaling pathway that is required for the internalization step of endocytosis. The Pkc1p kinase that is phosphorylated by Pkh1/2p kinases and plays a role in endocytosis was identified as one of the downstream effectors of this signaling cascade.

Bone anabolic effects of sonic/indian hedgehog are mediated by bmp-2/4-dependent pathways in the neonatal rat metatarsal model.

A neonatal rat metatarsal organ culture model has been employed to study the anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture system, a significant increase in endochondral ossification is measured by an increase in length of mineralized bone, after daily treatment for 7 days with Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH related protein (PTHrP) is a critical target of hedgehog function in endochondral ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-related increase in endochondral ossification is observed when metatarsals are treated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). However, this activity is not evident at higher doses of BMP-4 (200 ng/ml). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabolic action of Shh-N. This observation suggests that BMP-4 signaling can influence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossification is reversed by treatment with antisense oligonucleotides targeted against Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral ossification that mediates its effect via BMP-4 and Cbfa1-dependent pathways.

Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae.

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8-1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

Increased protein kinase or decreased PP2A activity bypasses sphingoid base requirement in endocytosis.

Lipids have been implicated in signal transduction and in several stages of membrane trafficking, but these two functions have not been functionally linked. In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. We show that inactivation of a protein phosphatase 2A (PP2A) or overexpression of one of two kinases, Yck2p or Pkc1p, can specifically suppress the sphingoid base synthesis requirement for endocytosis. The two kinases have an overlapping function because only a mutant with impaired function of both kinases is defective in endocytosis. An ultimate target of sphingoid base synthesis may be the actin cytoskeleton, because overexpression of the kinases and inactivation of PP2A substantially corrected the actin defect due to the absence of sphingoid base. These results suggest that sphingoid base controls protein phosphorylation, perhaps by activating a signal transduction pathway that is required for endocytosis and proper actin cytoskeleton organization in yeast.

Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo.

Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).

Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative effects of retinoids in Kaposi's sarcoma.

All-trans-retinoic acid (RA) is active in the treatment of Kaposi's sarcoma (KS), and retinoids inhibit KS cell growth in vitro. To understand the mechanism of retinoid action in KS, we studied the expression of autocrine growth factors of KS cells after RA treatment. We demonstrate that RA and its synthetic analogs inhibit the proliferation of KS cells by inhibiting the mRNA and protein levels of interleukin-6 (IL-6), an autocrine growth factor for KS cells. We further demonstrate that nuclear retinoid receptors (RA receptors [RARs] and retinoid X receptors [RXRs]) inhibit IL-6 promoter action by antagonizing the enhancer action of NF-IL6, a basic domain leucine zipper transcription factor belonging to the family of CAAT enhancer binding proteins. Furthermore, RARs and RXRs do not bind in vitro to an NF-IL6 binding site. However, the secondary folded structure of the DNA binding domain of RAR and RXR is obligatory for inhibiting NF-IL6 activity. Thus, NF-IL6 is a potential therapeutic target for the treatment of KS. Finally, using receptor-selective synthetic retinoids, we demonstrate that NF-IL6 antagonism and transactivation are separable functions of RAR alpha, thus indicating that synthetic retinoids with properties of NF-IL6 antagonism but lacking transactivation capabilities can be synthesized. Such retinoids might increase therapeutic potential in KS.

Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex.

In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.

Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts.

A specific terminal structure of preintegrative DNA is required for transposition of retroviruses and LTR-retrotransposons. We have used an anchored PCR technique to map the 3'ends of DNA intermediates synthesized inside yeast Ty1 and Ty3 retrotransposon virus-like particles. We find that, unlike retroviruses, Ty1 replicated DNA does not have two extra base pairs at its 3'ends. In contrast some Ty3 preintegrative DNA molecules have two extra nucleotides at the 3'end of upstream and downstream long terminal repeats. Moreover we find that some molecules of replicated Ty3 DNA have more than two extra nucleotides at the 3'end of the upstream LTR. This observation could be accounted for by imprecise RNAse H cutting of the PPT sequence. The site of Ty1 and Ty3 plus-strand strong-stop DNA termination was also examined. Our results confirm that the prominent Ty1 and Ty3 plus-strand strong-stop molecules harbor 12 tRNA templated bases but also show that some Ty1 and Ty3 plus-strand strong-stop DNA molecules harbor less tRNA templated bases. We propose that these less than full length plus-strand molecules could be active intermediates in Ty retrotransposon replication.

Negative regulation of two hyperproliferative keratinocyte differentiation markers by a retinoic acid receptor-specific retinoid: insight into the mechanism of retinoid action in psoriasis.

Retinoids down-regulate the expression of metalloproteinases, cytokines, and other genes involved in cell proliferation and inflammation. Tazarotene (AGN 190168), a retinoic acid receptor (RAR)-specific retinoid, is effective in the treatment of psoriasis, a hyperproliferative and inflammatory skin disease. Because negative regulation of genes appears to be important in the antiproliferative and antiinflammatory action of retinoids, we studied the down-regulation of genes in skin raft cultures by this antipsoriatic retinoid. By subtraction hybridization, we found that migration inhibitory factor-related protein (MRP-8) and skin-derived anti-leukoproteinase (SKALP) are down-regulated by AGN 190168. MRP-8 and SKALP are overexpressed in psoriatic lesions as compared to the normal epidermis, and they are markers of hyperproliferative keratinocyte differentiation. We also show that MRP-8 expression is retinoid inhibitable in cultured keratinocytes induced to differentiate with 10% serum or IFN-gamma, and that MRP-8 is inhibited by RAR but not by retinoid X receptor-specific retinoids in a dose-dependent manner. Finally, MRP-8, SKALP, and the previously characterized differentiation marker, transglutaminase I, are all down-regulated in vivo in psoriatic lesions after treatment with AGN 190168 in comparison to placebo. Taken together, these data suggest that these markers may be down-regulated by tazarotene in psoriasis through direct action on keratinocyte gene expression rather than by an overall tazarotene effect on lesional therapeutic status.

Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon.

Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.

Role of RNA primers in initiation of minus-strand and plus-strand DNA synthesis of the yeast retrotransposon Ty1.

The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae is a long terminal repeat mobile genetic element that transposes through an RNA intermediate. Initiation of minus-strand and plus-strand DNA synthesis are two critical steps during reverse transcription of the retrotransposon genome. Initiation of minus-strand DNA synthesis of the Ty1 element is primed by the cytoplasmic initiator methionine tRNA base paired to the primer binding site near the 5' end of the genomic RNA. A structural probing study of the primer tRNA-Ty1 RNA binary complex reveals that besides interactions between the primer binding site and the last 10 nucleotides at the 3' end of the primer tRNA, three short regions of Ty1 RNA named box 0, box 1 and box 2.1 interact with the T and D stems and loops of the primer tRNA. Some in vivo results underline the functional importance of the nucleotide sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primer tRNA play a role in the reverse transcription pathway. Plus-strand DNA synthesis is initiated from an RNase H resistant oligoribonucleotide spanning a purine-rich sequence, the polypurine tract (PPT). Two sites of initiation located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the TyB (pol) gene in the integrase coding sequence (PPT2) have been identified in the genome of Ty1. The two PPTs have an identical sequence, TGGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand DNA synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.

Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition.

Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.

RARs and RXRs: evidence for two autonomous transactivation functions (AF-1 and AF-2) and heterodimerization in vivo.

We have previously reported that the AB regions of retinoic acid receptors (RARs and RXRs) contain a transcriptional activation function capable of modulating the activity of the ligand-dependent activation function present in the C-terminal DE regions of these receptors. However, we could not demonstrate that these AB regions possess an autonomous activation function similar to the AF-1s found in the AB regions of steroid hormone receptors. Using the mouse CRBPII promoter as a reporter gene, we now report that the AB regions of RAR alpha, beta and gamma, as well as those of RXR alpha and gamma, contain an autonomous, ligand-independent activation function, AF-1, which can efficiently synergize with AF-2s. Moreover, AF-1s account for the ligand-independent, constitutive activation of transcription by RXR alpha and gamma. We also show that RARs and RXRs preferentially heterodimerize in solution in cultured cells in vivo, through the dimerization interface present in their E region, irrespective of the presence of all-trans or 9-cis retinoic acid. Furthermore, our results indicate that homodimeric interactions are not observed in cultured cells in vivo under conditions where heterodimeric interactions readily occur, which is in agreement with previous observations showing the preferential binding of RAR-RXR heterodimers to RA response elements in vitro.