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Biomedical subjects

Peter G Schultz

Publications and source records attributed to Peter G Schultz.

At least 73 records · Page 4Linked to original sources
PubMed2004

Small molecules that induce cardiomyogenesis in embryonic stem cells.

A phenotypic cell-based screen of a large combinatorial chemical library led to the identification of a class of diaminopyrimidine compounds (cardiogenol A-D) which can selectively and efficiently induce mouse embryonic stem cells (ESCs) to differentiate into cardiomyocytes. ESC-derived cardiomyocytes were shown to express multiple cardiac muscle markers, including myosin heavy chain, GATA-4, MEF2, and Nkx2.5, and spontaneously form beating regions. Such small molecules will serve as useful chemical probes to study cardiac muscle differentiation and may ultimately facilitate the therapeutic application of ESCs for cardiac repair.

Aniline Compounds↗
PubMed2004

Dedifferentiation of lineage-committed cells by a small molecule.

Combinatorial libraries were screened for molecules that induce mouse myogenic lineage committed cells to dedifferentiate in vitro. A 2,6-disubstituted purine, reversine, was discovered that induces lineage reversal of C2C12 cells to become multipotent progenitor cells which can redifferentiate into osteoblasts and adipocytes. This and other such molecules are likely to provide new insights into the molecular mechanisms that control cellular dedifferentiation and may ultimately be useful to in vivo stem cell biology and therapy.

Adipocytes↗
PubMed2004

Design, synthesis, and activity of HDAC inhibitors with a N-formyl hydroxylamine head group.

Histone deacetylases (HDAC) are promising targets for cancer chemotherapy. HDAC inhibitors are thought to act in part by disrupting normal cell cycle regulation, resulting in apoptosis and/or differentiation of transformed cells. Several HDAC inhibitors, which contain hydrophobic tails and the Zn(2+) chelator hydroxyamic acid as a head group, are potent inhibitors of HDACs both in vitro and in vivo. In this study, a related class of compounds with a N-formyl hydroxylamino head group has been synthesized and their ability to inhibit HDACs have been assayed in biochemical and cellular assays. These compounds were found to have comparable activities to suberoylanilide hydroxyamic acid (SAHA) in HDAC enzymatic assays and histone hyperacetylation cellular assays.

Drug Design↗
PubMed2004

RETRACTED: A new strategy for the synthesis of glycoproteins.

Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing beta-N-acetylglucosamine (GlcNAc)-serine at a defined position can be expressed in Escherichia coli in good yield and with high fidelity. The beta-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.

Acetylation↗
PubMed2004

A second-generation copper(II)-mediated metallo-DNA-base pair.

Metal-dependent pairing of nucleobases represents an alternative DNA base pairing scheme. Our first-generation copper(II)-mediated pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) metallo-base pair has a stability comparable to the natural base pairs dA:dT and dC:dG but does not have the selectivity of the Watson Crick base pairs. In order to increase the selectivity of base pair formation, a second-generation metallo-base pair was generated consisting of a pyridine-2,6-dicarboxamide (Dipam) and a pyridine (Py) nucleobase. This new metallo-base pair is more stable than the natural base pairs dA:dT and dC:dG and highly selective against mispairing. In addition, incorporation of multiple metallo-base pairs into DNA results in the formation of stable duplexes demonstrating that hydrogen bonding base pairs can efficiently be replaced by metal-dependent base pairs at multiple sites in DNA.

Base Pairing↗
PubMed2004

Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway.

Previously, a small molecule, purmorphamine, was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine, high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment, purmorphamine activates the Hedgehog (Hh) signaling pathway, resulting in the up- and downregulation of its downstream target genes, including Gli1 and Patched. Moreover, the known Hh signaling antagonists, cyclopamine and forskolin, completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling, and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease.

Alkaline Phosphatase↗
PubMed2004

Age-related effects of Ginkgo biloba extract on synaptic plasticity and excitability.

EGb 761 is a standardized extract from the Ginkgo biloba leaf and is purported to improve age-related memory impairment. The acute and chronic effect of EGb 761 on synaptic transmission and plasticity in hippocampal slices from young adult (8-12 weeks) and aged (18-24 months) C57Bl/6 mice was tested because hippocampal plasticity is believed to be a key component of memory. Acutely applied EGb 761 significantly increased neuronal excitability in slices from aged mice by reducing the population spike threshold and increased the early phase of long-term potentiation, though there was no effect in slices from young adults. In chronically treated mice fed for 30 days with an EGb 761-supplemented diet, EGb 761 significantly increased the population spike threshold and long-term potentiation in slices from aged animals, but had no effect on slices from young adults. The rapid effects of EGb 761 on plasticity indicate a direct interaction with the glutamatergic system and raise interesting implications with respect to a mechanism explaining its effect on cognitive enhancement in human subjects experiencing dementia.

Aging↗
PubMed2004

An expanding genetic code.

More than 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive and redox active amino acids, glycosylated and heavy atom derived amino acids in addition to those with keto, azido and acetylenic chains. In this article, we describe recent advances that make it possible to add new building blocks systematically to the genetic codes of bacteria, yeast and mammalian cells. Taken together these tools will enable the detailed investigation of protein structure and function, which is not possible with conventional mutagenesis. Moreover, by lifting the constraints of the existing 20-amino-acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.

Amino Acids↗
PubMed2004

A role for chemistry in stem cell biology.

Although stem cells hold considerable promise for the treatment of numerous diseases including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes and cancer, obstacles such as the control of stem cell fate, allogenic rejection and limited cell availability must be overcome before their therapeutic potential can be realized. This requires an improved understanding of the signaling pathways that affect stem cell fate. Cell-based phenotypic and pathway-specific screens of natural products and synthetic compounds have recently provided a number of small molecules that can be used to selectively control stem cell proliferation and differentiation. Examples include the selective induction of neurogenesis and cardiomyogenesis in murine embryonic stem cells, osteogenesis in mesenchymal stem cells and dedifferentiation in skeletal muscle cells. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicines for tissue repair and regeneration.

Animals↗
PubMed2004

Identification of a novel protein regulating microtubule stability through a chemical approach.

To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1, an NADP-dependent oxidoreductase. A role for NQO1 in influencing microtubule polymerization was confirmed through inhibition studies using known inhibitors and immunodepletion. Therefore, this chemical approach has identified a novel factor required for microtubule morphogenesis and cell division.

Amino Acid Sequence↗
PubMed2003

An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3' terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-kappaB signaling pathway.

Animals↗
PubMed2003

Site-specific incorporation of a redox-active amino acid into proteins.

The redox-active amino acid 3,4-dihydroxy-l-phenylalanine (DHP), which can undergo two-electron oxidation to a quinone, has been incorporated selectively and efficiently into proteins in Escherichia coli in response to a TAG codon. We have demonstrated that DHP can be oxidized electrochemically within the protein. The ability to incorporate a redox-active amino acid site specifically into proteins should facilitate the study of electron transfer in proteins, as well as enable the engineering of redox proteins with novel properties.

Amino Acyl-tRNA Synthetases↗
PubMed2003

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli.

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is 'orthogonal', meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA(Glu) sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.

Acylation↗
PubMed2003

Tom40 protein import channel binds to non-native proteins and prevents their aggregation.

Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

Cloning, Molecular↗
PubMed2003

Elucidating the molecular mechanism of cardiac remodeling using a comparative genomic approach.

It is proposed that analysis of global gene expression would provide an understanding of the molecular mechanisms of cardiac remodeling. However, previous studies have only provided "snapshots" of differential gene expression. Furthermore, the differences in gene expression between regions of the heart that can result in sampling variability have not been characterized. In this study, we employed the Affymetrix GeneChip technology to evaluate the patterns of expression in two different in vivo models of cardiac remodeling and in two different regions (left ventricle free wall and intraventricular septum) of the heart. Mice underwent transverse aortic constriction (TAC), myocardial infarction (MI), or sham operation, and RNA from the left ventricle free wall and the septum was isolated 1 wk later. Histological analysis showed profound myocyte hypertrophy and fibrosis in both the septum and the left ventricle free wall of the TAC model, whereas, in the MI model, only the left ventricle exhibited hypertrophy. These differences were also reflected in the expression analysis. In conclusion, our analysis shows that regional differences in gene expression exist in the heart. Moreover, common pathways that are coregulated in both models exist, and these might be central to the hypertrophic phenotype regardless of the initial hypertrophic stimuli.

Animals↗
PubMed2003

One-pot two-step microwave-assisted reaction in constructing 4,5-disubstituted pyrazolopyrimidines.

[reaction: see text] A microwave-assisted reaction was developed to facilitate the construction of 4,5-disubstituted pyrazolopyrimidines. This one-pot two-step process involves a sequential S(N)Ar displacement of the C4 chloro substituent with various anilines and amines, followed by a Suzuki coupling reaction with different boronic acids. Using microwave irradiation leads to high product conversion, low side product formation, and shorter reactions.

Journal Article↗
PubMed2003

Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae.

Using a novel genetic selection, we have identified a series of mutants of the E. coli tyrosyl-tRNA synthetase that selectively charge an amber suppressor tRNA with p-(propargyloxy)phenylalanine and p-azidophenylalanine in yeast. These evolved tRNA-synthetase pairs can be used to site-specifically label proteins with functional groups orthogonal to normal biological chemistries. As an example, we have shown that proteins containing these amino acids can be efficiently bioconjugated with small organic molecules by a [3 + 2] cycloaddition reaction that is mild enough for the manipulation of biological samples.

Acetylene↗