Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors

Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.     

Physiological basis of reduced Al tolerance in ditelosomic lines of Chinese Spring wheat

Aluminum tolerance was assessed in the moderately Al-tolerant wheat (Triticum aestivum L.) cultivar Chinese Spring and a set of ditelosomic lines derived from Chinese Spring. Three ditelosomic lines lacking chromosome arms 4DL, 5AS and 7AS, respectively, exhibited decreased Al tolerance relative to the euploid parent Chinese Spring based on reduced root growth in Al-containing solutions. The physiological basis of the reduced Al tolerance was investigated. Measurements by inductively coupled argon plasma mass spectroscopy of root apical Al accumulation demonstrated that two of these three lines had a decreased ability to exclude Al from the root apex, the site of Al phytotoxicity. As Al-induced malate exudation has been suggested to be an important physiological mechanism of Al tolerance in wheat, this parameter was quantified and malate exudation was shown to be smaller in all three deletion lines compared with Chinese Spring. These results suggest that the decreased Al tolerance in at least two of the three ditelosomic lines is due to the loss of different genes independently influencing a single Al-tolerance mechanism, rather than to the loss of genes encoding alternative Al-tolerance mechanisms. Received: 3 July 2000 / Accepted: 9 August 2000     

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.     

Targeting the immune system: novel therapeutic approaches in squamous cell carcinoma of the head and neck

Despite advances in surgery, radiotherapy, and chemotherapy, the overall survival rates for patients with squamous cell carcinoma of the head and neck (SCCHN) have not changed over the last decades. Clearly, novel therapeutic strategies are needed for this cancer, which is highly immunosuppressive. Therefore, biologic therapies able to induce and/or up-regulate antitumor immune responses could represent a complementary approach to conventional treatments. Because patients with SCCHN are frequently immunocompromised due to the elimination or dysfunction of critical effector cells of the immune system, it might be necessary to restore these immune functions to allow for the generation of more effective antitumor host responses. Simultaneously, to prevent tumor escape, it might be necessary to alter attributes of the malignant cells. The present review summarizes recent advances in the field of immunotherapy of SCCHN, including techniques of nonspecific immune stimulation, the use of monoclonal antibodies, advances in adoptive immunotherapy and genetic engineering, as well as anticancer vaccines. These biologic therapies, alone or in combination with conventional treatment, are likely to develop into useful future treatment options for patients with SCCHN.     

Macrocyclic piperazinones as potent dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I

A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.     

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.     

Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus

Although vascular endothelial growth factor (VEGF) plays a vital role in lung vascular growth in the embryo, its role in maintaining endothelial function and modulating vascular structure during late fetal life has not been studied. We hypothesized that impaired lung VEGF signaling causes pulmonary hypertension, endothelial dysfunction, and structural remodeling before birth. To determine whether lung VEGF expression is decreased in an experimental model of persistent pulmonary hypertension of the newborn (PPHN), we measured lung VEGF and VEGF receptor protein content from fetal lambs 7-10 days after ductus arteriosus ligation (132-140 days gestation; term = 147 days). In contrast with the surge in lung VEGF expression during late gestation in controls, chronic intrauterine pulmonary hypertension reduced lung VEGF expression by 78%. To determine whether VEGF inhibition during late gestation causes pulmonary hypertension, we treated fetal lambs with EYE001, an aptamer that specifically inhibits VEGF(165). Compared with vehicle controls, EYE001 treatment elevated pulmonary artery pressure and pulmonary vascular resistance by 22 and 50%, respectively, caused right ventricular hypertrophy, and increased wall thickness of small pulmonary arteries. EYE001 treatment reduced lung endothelial nitric oxide synthase protein content by 50% and preferentially impaired the pulmonary vasodilator response to ACh, an endothelium-dependent agent. We conclude that chronic intrauterine pulmonary hypertension markedly decreases lung VEGF expression and that selective inhibition of VEGF(165) mimics the structural and physiological changes of experimental PPHN. We speculate that hypertension downregulates VEGF expression in the developing lung and that impaired VEGF signaling may contribute to the pathogenesis of PPHN.     

Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of (125)I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol. min(-1). 10(6) cells(-1). Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-beta-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased (125)I-albumin uptake 2.3-fold. At 37 degrees C, (125)I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC(50) = 1 microM). Using a two-site model, we estimated by Scatchard analysis the affinity (K(D)) and maximal capacity (B(max)) of albumin uptake to be 0.87 microM (K(D1)) and 0.47 pmol/10(6) cells (B(max1)) and 93.3 microM (K(D2)) and 20.2 pmol/10(6) cells (B(max2)). At 4 degrees C, we also observed two populations of specific binding sites, with high (K(D1) = 13.5 nM, 1% of the total) and low (K(D2) = 1.6 microM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.