Differential association of HLA-B*2705 and B*2709 to ankylosing spondylitis correlates with limited peptide subsets but not with altered cell surface stability

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to beta2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.     

Genetic mapping of the whirler mutation

The whirler (wi) mutation on mouse Chromosome (Chr) 4 results in an autosomal recessive neuroepithelial deafness and vestibular dysfunction exhibited as a characteristic shaker-waltzer behavior (deafness, circling, and head-bobbing). We have constructed a genetic linkage map across the wi region in both an interspecific [(wi/wi× CAST/Ei)F₁×wi/wi] backcross (n = 817) and an intraspecific [(wi/wi× CBA/Ca)F₁×wi/wi)] backcross (n = 335). In the interspecific backcross, wi was found to be non-recombinant with Orm1, 0.12 cM distal of D4Mit87 and Ambp, and 0.12 cM proximal of CD301. In the intraspecific backcross, wi was found to be non-recombinant with Orm1 and D4Mit244, 0.3 cM distal of Mup1, and 0.6 cM proximal of Tnc. We also report a family from the interspecific backcross that shows evidence of multiple recombinations across the region of mouse Chr 4 around the wi locus. These rearrangements appear specific to both the region and the family. Received: 10 July 1998 / Accepted: 19 January 1999     

IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.     

The enlargement of the hormone immune deprivation concept to the blocking of TGFα-autocrine loop: EGFR signaling inhibition

Transforming growth factor alpha (TGFα) is a potent ligand of the epidermal growth factor receptor (EGFR). EGFR is frequently over-expressed in epithelial tumors and endogenous ligands, mostly TGFα, are frequently co-expressed with EGFR, potentially resulting in autocrine stimulation of tumor cell growth. Therefore, different therapeutic approaches aim for the inactivation of TGFα/EGF/EGFR signaling system, but no approach is based on TGFα as a target. The principal goal of this work was to assess the potential of an active specific immunotherapy approach to block the TGFα/EGFR autocrine loop. For the proof of the concept, a fusion protein between human TGFα (hTGFα) and P64k protein from Neisseria meningitidis was generated, and its immunogenicity characterized in a mouse model using different adjuvants. All immunogens were effective for the generation of specific humoral responses against hTGFα. The inmunodominant epitope of hTGFα when immunizing mice with the fusion protein involved the C-loop/C-terminal region. This region includes key residues for hTGFα binding to EGFR. The anti-hTGFα immune mice sera recognized the natural hTGFα precursor in A431 cells and hTGFα-transfected 3T3 fibroblasts as revealed by flow cytometry analysis and immunoblotting. They inhibited the binding of ¹²⁵I-TGFα to the EGFR, EGFR-autophosphorylation, and downstream activation of MAP kinases as well as proliferation of two EGFR-expressing human carcinoma cell lines. These data suggest that EGFR signaling activation by the hTGFα autocrine loop may be inhibited in vivo by induction of specifically blocking antibodies. The fusion protein reported in this paper could be a potential immunogen for the development of a new cancer vaccine. Part of this work was supported by a travel scholarship sponsored by the Boehringer Ingelheim Fonds     

The extreme plant-growth-promoting properties of Pantoea phytobeneficialis MSR2 revealed by functional and genomic analysis

Numerous Pantoea strains are important because of the benefit they provide in the facilitation of plant growth. However, Pantoea have a high level of genotypic diversity and not much is understood regarding their ability to function in a plant beneficial manner. In the work reported here, the plant growth promotion activities and the genomic properties of the unusual Pantoea phytobeneficialis MSR2 are elaborated, emphasizing the genetic mechanisms involved in plant colonization and growth promotion. Detailed analysis revealed that strain MSR2 belongs to a rare group of Pantoea strains possessing an astonishing number of plant growth promotion genes, including those involved in nitrogen fixation, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid deaminase activity, indoleacetic acid and cytokinin biosynthesis, and jasmonic acid metabolism. Moreover, the genome of this bacterium also contains genes involved in the metabolism of lignin and other plant cell wall compounds, quorum-sensing mechanisms, metabolism of plant root exudates, bacterial attachment to plant surfaces and resistance to plant defences. Importantly, the analysis revealed that most of these genes are present on accessory plasmids that are found within a small subset of Pantoea genomes, reinforcing the idea that Pantoea evolution is largely mediated by plasmids, providing new insights into the evolution of beneficial plant-associated Pantoea.     

Genetic Diversity of <Emphasis Type="Italic">Cercospora kikuchii</Emphasis> Isolates From Soybean Cultured in Argentina as Revealed by Molecular Markers and Cercosporin Production

Leaf blight and purple seed, caused by the fungal pathogen Cercospora kikuchii (Matsumoto & Tomoyasu) M. W. Gardner are very important diseases of soybean (Glycine max L. Merr.) in Argentina. The aims of this work were: (a) to confirm and to assess the genetic variability among C. kikuchii isolates collected from different soybean growing areas in Santa Fe province using inter simple sequence repeats (ISSR) markers and sequence information from the internal transcribed spacer (ITS) region of rDNA and (b) to analyze the cercosporin production of the regional C. kikuchi isolates in order to assess whether there was any relationship between the molecular profiles and the toxin production. Isolates from different regions in Santa Fe province were studied. The sequence of the ITS regions showed high similarity (99–100%) to the GenBank sequences of C. kikuchii BRCK179 (accession number AY633838). The ISSR markers clustered all the isolates into many groups and cercosporin content was highly variable among isolates. No relationship was observed between ITS region, ISSR groups and origin or cercosporin content. The high degree of genetic variability and cercosporin production among isolates compared in this study characterizes a diverse population of C. kikuchii in the region.     

Neonatal astrocyte damage is sufficient to trigger progressive striatal degeneration in a rat model of glutaric acidemia-I

Background

We have investigated whether an acute metabolic damage to astrocytes during the neonatal period may critically disrupt subsequent brain development, leading to neurodevelopmental disorders. Astrocytes are vulnerable to glutaric acid (GA), a dicarboxylic acid that accumulates in millimolar concentrations in Glutaric Acidemia I (GA-I), an inherited neurometabolic childhood disease characterized by degeneration of striatal neurons. While GA induces astrocyte mitochondrial dysfunction, oxidative stress and subsequent increased proliferation, it is presently unknown whether such astrocytic dysfunction is sufficient to trigger striatal neuronal loss.

Methodology/Principal Findings

A single intracerebroventricular dose of GA was administered to rat pups at postnatal day 0 (P0) to induce an acute, transient rise of GA levels in the central nervous system (CNS). GA administration potently elicited proliferation of astrocytes expressing S100β followed by GFAP astrocytosis and nitrotyrosine staining lasting until P45. Remarkably, GA did not induce acute neuronal loss assessed by FluoroJade C and NeuN cell count. Instead, neuronal death appeared several days after GA treatment and progressively increased until P45, suggesting a delayed onset of striatal degeneration. The axonal bundles perforating the striatum were disorganized following GA administration. In cell cultures, GA did not affect survival of either striatal astrocytes or neurons, even at high concentrations. However, astrocytes activated by a short exposure to GA caused neuronal death through the production of soluble factors. Iron porphyrin antioxidants prevented GA-induced astrocyte proliferation and striatal degeneration in vivo, as well as astrocyte-mediated neuronal loss in vitro.

Conclusions/Significance

Taken together, these results indicate that a transient metabolic insult with GA induces long lasting phenotypic changes in astrocytes that cause them to promote striatal neuronal death. Pharmacological protection of astrocytes with antioxidants during encephalopatic crisis may prevent astrocyte dysfunction and the ineluctable progression of disease in children with GA-I.     

Genetic transformation of <Emphasis Type="Italic">Solanum chrysotrichum</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the production of antifungal saponins

Solanum chrysotrichum (Solanaceae) synthesizes a family of six antifungal spirostanol saponins designated as SC-1 to SC-6. The production of saponins by wild-type plants is variable depending on the environmental conditions. In order to develop an in vitro system for the sustained production of these saponins, transformed cell suspension cultures of S. chrysotrichum were established from nodal explants of 3-mo-old plantlets by infecting with the Agrobacterium tumefaciens strain C58/pBI12. From these cultures, kanamycin-resistant and phytohormone-independent cell suspension line C58 5.1.1 was obtained. PCR and Southern blot analyses were used to confirm the integration of the wild-type T-DNA into the plant genome. Batch cultures of the C58 5.1.1 cell line were grown in phytohormone-free MS liquid medium for 25 d. First-order growth kinetics and the production of the antifungal saponins (SC-2, SC-3, and SC-4) were determined by dry weight and quantified by HPLC, respectively, from the cells as well as the culture medium. Based on the cell biomass, the specific growth rate was 0.09 d⁻¹ and the yield of SC-2 reached 5.5% of dry weight, representing 40 times higher amount than that produced in plant leaves. SC-3 was recovered with a maximum yield of 0.9% of dry weight, whereas SC-4 was accumulated at 1.1% of dry weight. Saponins SC-2 and SC-3 were also excreted into the culture medium in low concentrations.