Structure-property relationship of donor-acceptor acridones - an optical, electrochemical and computational study.

The synthesis of two neutral acridone derivatives was carried out to design media sensitive chromophores by taking advantage of intramolecular charge transfer (ICT) features. The molecules comprised two different donor-acceptor-donor triads, with absorption maxima at 425 nm and 520 nm, for the ketone and dicyanomethylene derivatives, respectively. The ketone variant exhibited fluorescence at room temperature, whereas the dicyanomethylene derivative was only emissive in frozen hexane. The ketone emission was highly solvatochromic, with Stokes shifts that ranged from 5000 cm(-1) to 10 000 cm(-1). Electrochemically, both compounds displayed similar oxidation potentials at approximately 0.35 V versus ferrocene/ferrocenium, which was anticipated since both systems employ the same ethynylaniline donor portion of the molecule, whereas only the 9-dicyanomethylene derivative showed a reduction peak at -1.5 V vs. Fc/Fc(+). Additional spectroelectro-chemical experiments supported a delocalized cationic charge on the ethynylaniline fragments during oxidation and that during electrochemical reduction the dicyanomethylene moieties show localized anionic charge. All of the experimental observations are finally compared to DFT, TDDFT and NICS(0) computations to gain insight into the transitions involved and deduce the role of the acridone core in stabilizing its oxidized and reduced forms.

Cis-acting regulation of brain-specific ANK3 gene expression by a genetic variant associated with bipolar disorder.

Several genome-wide association studies for bipolar disorder (BD) have found a strong association of the Ankyrin 3 (ANK3) gene. This association spans numerous linked single-nucleotide polymorphisms (SNPs) in an ~250-kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of the six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as Ankyrin-G. Using RNA ligase-mediated rapid amplification of cDNA ends to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites and coupling of specific 5' ends with 3' mRNA splicing events in postmortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD-associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 that may be relevant to BD pathophysiology.

Tyrosine phosphorylation of tub and its association with Src homology 2 domain-containing proteins implicate tub in intracellular signaling by insulin.

A mutation in the tub gene leads to maturity-onset obesity, insulin resistance, and progressive retinal and cochlear degeneration in mice. tub is a member of a growing family of genes that encode proteins of unknown function that are remarkably conserved across species. The absence of obvious transmembrane domain(s) or signal sequence peptide motif(s) suggests that Tub is an intracellular protein. Additional sequence analysis revealed the presence of putative tyrosine phosphorylation motifs and Src homology 2 (SH2)-binding sites. Here we demonstrate that in CHO-IR cells, transfected Tub is phosphorylated on tyrosine in response to insulin and insulin-like growth factor-1 and that in PC12 cells, insulin but not EGF induced tyrosine phosphorylation of endogenous Tub. In vitro, Tub is phosphorylated by purified insulin receptor kinase as well as by Abl and JAK 2 but not by epidermal growth factor receptor and Src kinases. Furthermore, upon tyrosine phosphorylation, Tub associated selectively with the SH2 domains of Abl, Lck, and the C-terminal SH2 domain of phospholipase Cgamma and insulin enhanced the association of Tub with endogenous phospholipase Cgamma in CHO-IR cells. These data suggest that Tub may function as an adaptor protein linking the insulin receptor, and possibly other protein-tyrosine kinases, to SH2-containing proteins.

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor.

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.

Polymorphism in a ferritin H gene from chromosome 6p.

This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.

Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene.

We have purified a minor extracellular serine protease from Bacillus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.

Identification of two human ferritin H genes on the short arm of chromosome 6.

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.