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Biomedical subjects

J Chmielewski

Publications and source records attributed to J Chmielewski.

At least 19 recordsLinked to original sources
PubMed2006

Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing.

The effect of water- and steam-cooking on the content of vitamin C, polyphenols, carotenoids, tocopherols and glucosinolates, as well as on the antioxidant activity of broccoli, are reported. Flavonoids, phenolic acids, vitamins C and E, beta-carotene, lutein, and glucosinolates in domestically processed broccoli were quantified using high-performance liquid chromatography (HPLC) methods; total polyphenols were determined with Folin-Ciocalteu reagent. The antioxidant capacities of broccoli extracts were evaluated using the Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The results indicated that steam-cooking of broccoli results in an increase in polyphenols, as well as the main glucosinolates and their total content as compared with fresh broccoli, whereas cooking in water has the opposite effect. Steam-cooking of broccoli has no influence on vitamin C, whereas cooking in water significantly lowers its content. Both, water- and steam-cooking of broccoli results in an increase in beta-carotene, lutein, and alpha- and gamma-tocopherols as compared with fresh broccoli. Similar effects of steaming and water-cooking of broccoli on their antioxidant activity were observed.

Antioxidants↗
PubMed2005

Liposomal cargo unloading induced by pH-sensitive peptides.

Amphiphilic peptides designed to have a pH-dependent conformational change and membrane activity are described. At physiologic pH, the peptides would exist in a random coil conformation, but at endosomal pH values they would switch to amphiphilic alpha-helices, disrupt membranes, and release liposomal contents. A series of peptides have been investigated that contain a high percentage of Glu residues for the pH-induced conformational switch, and Leu residues for optimal lipid binding. Circular dichroism (CD) results in aqueous and liposomal environments were performed and demonstrate a pH-dependent shift to helicity upon acidification. Liposomal release data at neutral and acidic pH, also document the success of this design strategy.

Binding Sites↗
PubMed2004

Probing essential residues for cellular uptake with a cationic nuclear localization signal sequence.

The nuclear localization signal sequence (NLS) of the transcription factor NF-kappaB is a cationic peptide with the ability to cross the cytoplasmic membrane and facilitate the delivery of attached cargo, such as DNA and proteins, to cells. Previous research had pointed to the essential role of cationic residues, therefore, the importance of residues within the NLS of NF-kappaB was evaluated for cellular uptake using an alanine replacement strategy. Although it was expected that removal of the cationic groups would have the greatest effect on membrane translocation, the most significant decreases in cellular uptake occurred with the replacement of the hydrophilic Q6 (80%) and the hydrophobic L8 (70%) residues. Replacement of the positively charged residues resulted in 30-40% decrease in cellular uptake, indicating that electrostatic interactions are not the primary driving force for membrane translocation.

Active Transport, Cell Nucleus↗
PubMed2001

The design of self-replicating helical peptides.

The self-assembly of helical peptides and information transfer through autocatalysis and cross-catalysis are the foundation of peptide-based molecular evolution models. Many fundamental properties of living systems, such as environmental sensitivity, chiroselectivity, cross-catalysis, dynamic error correction and conditional selection, are exhibited by various self-replicating peptide systems. Recently, advances have been made in the design of peptide systems with autocatalytic and cross-catalytic properties.

Amino Acid Sequence↗
PubMed2001

DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47.

The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6 x 10(-9) M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.

Amino Acid Sequence↗
PubMed2001

Alpha-lactose monohydrate single crystals as hosts for matrix isolation of guest biopolymers.

Single crystals of alpha-lactose monohydrate show a remarkable tendency to include biopolymers, such as proteins, oligonucleotides and dextrans, within the growing lattice. Glycosylation increased the amount of protein contained within the crystals. The guest molecules were found only within the (010) growth sector of the hatchet shaped crystals, thereby binding preferentially to one of the seven developed crystal faces. The topographical features of the active surface are described.

Animals↗
PubMed1999

Probing the role of interfacial residues in a dimerization inhibitor of HIV-1 protease.

The importance of each side chain of a cross-linked interfacial peptide inhibitor of HIV-1 protease was evaluated using an alanine scanning approach. Whereas the parent inhibitor has an IC50 value of 350 nM, values for the mutations reported here range from 280-9200 nM. The relative importance or each residue was thus assigned and correlated to the solvent accessible surface area (SASA) exposed upon mutation.

Alanine↗
PubMed1999

A pH-tunable peptide ligase.

A chemical ligation system is reported, in which a highly acidic coiled-coil peptide was used to template two basic peptide fragments and catalyze their condensation, in a pH-tunable fashion, to generate a coiled-coil product. This template showed a high catalytic efficiency (with single turnover) under neutral conditions. Under acidic conditions, however, its catalytic efficiency was reduced by approximately 4500-fold.

Amino Acid Sequence↗
PubMed1999

Structure-function relationship in a beta-sheet peptide inhibitor of E47 dimerization and DNA binding.

A beta-sheet peptide inhibitor, 2H10, has been developed that inhibits the dimerization of the transcription factor E47. Inhibition of E47 dimerization has been demonstrated to also inhibit the DNA binding of this transcription factor. Truncated peptides based on 2H10 have demonstrated that the beta-sheet content of these peptides directly correlates with their inhibitory properties. Individual residues within 2H10 were identified that were responsible for the beta-sheet secondary structure by employing an alanine replacement strategy. The beta-sheet character of the alanine mutants also correlated well with their inhibition of E47 DNA binding. These results provide further evidence that interactions between the interfacial peptide inhibitors of E47 and the transcription factor itself are mediated by a beta-sheet structure.

Amino Acid Sequence↗
PubMed1999

Helical peptide and protein design.

The design of dimeric coiled-coils has ultimately led to novel applications, such as self-replicating peptide systems, whereas the structural features of the less common trimeric coiled-coil continue to be elucidated. Novel topologies have been discovered in designed proteins, as exemplified by the right-handed tetrameric coiled-coil and the inverted U four-helix bundle, and a single switch of two amino acids within a protein has been shown to be sufficient to designate a new protein fold. Conformational switching from helix to sheet has been observed for designed peptides and transcription factors, whereas peptides designed from beta-amino acids have been found to adopt a helical conformation in aqueous solution.

Amino Acid Sequence↗
PubMed1998

Selective amplification by auto- and cross-catalysis in a replicating peptide system.

Self-replication has been demonstrated in synthetic chemical systems based on oligonucleotides, peptides and complementary molecules without natural analogues. However, within a living cell virtually no molecule catalyses its own formation, and the search for chemical systems in which both auto- and cross-catalysis can occur has therefore attracted wide interest. One such system, consisting of two self-replicating peptides that catalyse each other's production, has been reported. Here we describe a four-component peptide system that is capable of auto- and cross-catalysis and allows for the selective amplification of one or more of the products by changing the reaction conditions. The ability of this system selectively to amplify one or more molecules in response to changes in environmental conditions such as pH or salt concentration supports the suggestions that self-replicating peptides may have played a role in the origin of life.

Amino Acid Sequence↗
PubMed1998

Endothiopeptide inhibitors of HIV-1 protease.

Endothiopeptide inhibitors of HIV-1 protease were synthesized by chemical and enzymatic methods to individually replace each backbone amide bond in 1 with a thioamide-linkage. Interestingly, agent 7, which contains a thioamide-linkage between the P2' and P3' positions of 1, was the most potent, competitive inhibitor of HIV-1 protease with a Ki of 3.4 microM.

Chromatography, High Pressure Liquid↗
PubMed1998

Uncoiling c-Jun coiled coils: inhibitory effects of truncated Fos peptides on Jun dimerization and DNA binding in vitro.

c-Jun is an oncoprotein that comprises a portion of the AP-1 transcription factor and belongs to the basic-leucine zipper (bZIP) DNA binding protein family. Using peptides derived from the leucine zipper region of Fos, we have developed agents that inhibit Jun's DNA binding in the low micromolar range. Fos peptides that were effective inhibitors in the DNA binding assay were also found to inhibit cellular Jun binding to an AP-1 site in a luciferase reporter plasmid in MCF-7 cells. Size exclusion studies confirmed that peptides that inhibit the DNA binding of Jun also inhibit its dimerization. These peptides were found to have a cytotoxic effect on the MCF-7 cell line when delivered with the transfecting agent Tfx-50, possibly due to their role as transcription factor regulators.

Amino Acid Sequence↗
PubMed1998

A beta-sheet peptide inhibitor of E47 dimerization and DNA binding.

BACKGROUND: Many transcription factors are active only in their dimeric form, including the basic-helix-loop-helix (bHLH) family of transcription factors. The disruption of the dimer therefore presents a means of inhibiting the biological functions of such transcription factors. E47 is a homodimeric bHLH transcription factor with a four-helix bundle dimerization interface. Here, we investigate the concept of dimerization inhibition using peptides derived from the dimerization domain of E47. RESULTS: We have synthesized several peptides corresponding to the E47 dimerization interface that inhibit E47 DNA-binding activity with IC50 values in the range of 3.6-120 mM. Interestingly, helix II; a peptide corresponding to the carboxy-terminal helix of the E47 dimerization interface, adopted a beta-sheet structure in solution, as shown using circular dichroism (CD), and inhibited the binding of E47 to DNA at equimolar concentrations. Size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments verified that this peptide prevented E47 dimerization. Furthermore, CD experiments provided evidence that helix II could induce a beta-sheet secondary structure upon the highly alpha-helical E47 bHLH domain. CONCLUSIONS: This study is the first demonstration of dissociative inhibition in the bHLH class of transcription factors and also provides an example of beta-sheet induction in an alpha-helical protein. Future experiments will prove the structural determinants of the beta-sheet secondary structure in helix II and investigate the generality of the dissociative strategy in other transcription factor families.

Amino Acid Sequence↗
PubMed1998

Inhibiting the dimeric restriction endonuclease EcoRI using interfacial helical peptides.

BACKGROUND: Many enzymes are active only in a dimeric form, including a variety of type II restriction endonucleases. Disruption of subunit interactions is therefore a potential method for multimeric enzyme inhibition. EcoRI is a homodimeric restriction endonuclease, the dimeric interface of which consists of a four-helix bundle. We set out to design helical peptides to interact with this interface and block dimer formation, thus rendering EcoRI inactive. RESULTS: Here we describe two synthetic, helical peptides based on the interfacial region of EcoRI. Both peptides inhibit the enzyme, but the peptide derived from the alpha 4 helix of EcoRI had both a higher helical content and better efficacy than a variant peptide, alpha 4(Leu), that has three Ile-->Leu mutations (IC50 values of 27 microM and 90 microM, and helical contents of 29% and 10%, respectively). Size-exclusion chromatography confirmed that the alpha 4 peptide disrupted dimerization of EcoRI, and circular dichroism indicated that EcoRI remained folded upon binding to alpha 4. Inhibition with alpha 4 and alpha 4(Leu) was shown to be specific for EcoRI, as the dimeric restriction enzyme PvuII was not affected by the peptides. CONCLUSIONS: Interfacial peptide inhibitors of the dimeric EcoRI were obtained that both inhibit dimerization and endonuclease activity. The peptide sequence with a preference for a helical conformation was a more effective inhibitor, presumably because the more preorganized state enhanced interactions with the helical interface of EcoRI. The specific nature of this endonuclease-peptide interaction was also confirmed. The potential of this strategy for inhibiting other enzyme classes is currently being addressed.

Chromatography, Gel↗