Synthesis and preliminary pharmacological evaluation of novel derivatives of L-beta-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT3

A series of beta-benzylaspartate derivatives were prepared from N-trityl-L-aspartate dimethyl ester and evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure-activity studies suggests that the position occupied by the aromatic ring of beta-benzylaspartate within the binding site of EAAT3 may be different from that occupied by comparable groups in previously identified inhibitors, such as L-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-position of the aromatic ring of beta-benzylaspartate can increase the potency with which the analogues inhibit EAAT3.     

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.     

Nuclear and cpDNA sequences combined provide strong inference of higher phylogenetic relationships in the phlox family (Polemoniaceae)

Members of the phlox family (Polemoniaceae) serve as useful models for studying various evolutionary and biological processes. Despite its biological importance, no family-wide phylogenetic estimate based on multiple DNA regions with complete generic sampling is available. Here, we analyze one nuclear and five chloroplast DNA sequence regions (nuclear ITS, chloroplast matK, trnL intron plus trnL-trnF intergeneric spacer, and the trnS-trnG, trnD-trnT, and psbM-trnD intergenic spacers) using parsimony and Bayesian methods, as well as assessments of congruence and long branch attraction, to explore phylogenetic relationships among 84 ingroup species representing all currently recognized Polemoniaceae genera. Relationships inferred from the ITS and concatenated chloroplast regions are similar overall. A combined analysis provides strong support for the monophyly of Polemoniaceae and subfamilies Acanthogilioideae, Cobaeoideae, and Polemonioideae. Relationships among subfamilies, and thus for the precise root of Polemoniaceae, remain poorly supported. Within the largest subfamily, Polemonioideae, four clades corresponding to tribes Polemonieae, Phlocideae, Gilieae, and Loeselieae receive strong support. The monogeneric Polemonieae appears sister to Phlocideae. Relationships within Polemonieae, Phlocideae, and Gilieae are mostly consistent between analyses and data permutations. Many relationships within Loeselieae remain uncertain. Overall, inferred phylogenetic relationships support a higher-level classification for Polemoniaceae proposed in 2000.     

Matrix deposition of tryptophan-containing allelic variants of type IX collagen in developing human cartilage.

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the alpha2 (Trp2) or alpha3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the alpha3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its alpha2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix.     

Cerebro-Oculo-Facio-Skeletal Syndrome with a Nucleotide Excision–Repair Defect and a Mutated XPD Gene, with Prenatal Diagnosis in a Triplet Pregnancy

Cerebro-oculo-facio-skeletal (COFS) syndrome is a recessively inherited rapidly progressive neurologic disorder leading to brain atrophy, with calcifications, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low to normal birth weight, growth failure, brain dysmyelination with calcium deposits, cutaneous photosensitivity, pigmentary retinopathy and/or cataracts, and sensorineural hearing loss. Cultured CS cells are hypersensitive to UV radiation, because of impaired nucleotide-excision repair (NER) of UV-induced damage in actively transcribed DNA, whereas global genome NER is unaffected. The abnormalities in CS are caused by mutated CSA or CSB genes. Another class of patients with CS symptoms have mutations in the XPB, XPD, or XPG genes, which result in UV hypersensitivity as well as defective global NER; such patients may concurrently have clinical features of another NER syndrome, xeroderma pigmentosum (XP). Clinically observed similarities between COFS syndrome and CS have been followed by discoveries of cases of COFS syndrome that are associated with mutations in the XPG and CSB genes. Here we report the first involvement of the XPD gene in a new case of UV-sensitive COFS syndrome, with heterozygous substitutions-a R616W null mutation (previously seen in patients in XP complementation group D) and a unique D681N mutation-demonstrating that a third gene can be involved in COFS syndrome. We propose that COFS syndrome be included within the already known spectrum of NER disorders: XP, CS, and trichothiodystrophy. We predict that future patients with COFS syndrome will be found to have mutations in the CSA or XPB genes, and we document successful use of DNA repair for prenatal diagnosis in triplet and singleton pregnancies at risk for COFS syndrome. This result strongly underlines the need for screening of patients with COFS syndrome, for either UV sensitivity or DNA-repair abnormalities.     

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.