Biomedical subjects

H F Hansen

Publications and source records attributed to H F Hansen.

At least 19 recordsLinked to original sources

PubMed2004

Effect of LNA modifications on small molecule binding to nucleic acids.

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.

Bisbenzimidazole↗

PubMed2001

PNA synthesis using a novel Boc/acyl protecting group strategy.

The synthesis of novel Boc/acyl protected monomers for the synthesis of peptide nucleic acid (PNA) is described. The oligomerization protocol using these new monomers has been optimized with regard to coupling reagents. The use of base-labile acyl protecting groups at the exocyclic amines of the heterocyclic bases (isobutyryl for guanine and benzoyl for adenine and cytosine) and a PAM-linked solid support offers an attractive alternative to the present procedures used in PNA synthesis. This strategy has been applied for the synthesis of a test 17mer PNA on both control pore glass (CPG) and a polystyrene MBHA support and was used in the preparation of PNA-DNA chimeras.

Biochemistry↗

PubMed2001

Linker length in podophyllotoxin-acridine conjugates determines potency in vivo and in vitro as well as specificity against MDR cell lines.

We have synthesized two podophyllotoxin-acridine conjugates-pACR6 and pACR8. In these compounds an 9-acridinyl moiety is beta linked to the C4 carbon of the four ring system in 4'-demethylepipodophyllotoxin (epiDPT) via eighter an N-6-aminohexanylamide linker (pACR6) or via an N-8-aminooctanylamide linker containing two more carbon atoms (pACR8). The acridine-linker moiety occupies the position where different glucoside moieties, dispensable for activity, are normally linked to epiDPT in the well known epipodophyllotoxins VP-16 and VM-26. As with VP-16 and VM-26, pACR6 and pACR8 show evidence of being topoisomerase II poisons as they stimulate topoisomerase II mediated DNA cleavage in vitro and induce DNA damage in vivo. This in vivo DNA damage, as well as pACR6/pACR8 mediated cytotoxicity, is antagonized by the catalytic topoisomerase II inhibitors ICRF-187 and aclarubicin, demonstrating that topoisomerase II is a functional biological target for these drugs. Despite their structural similarities, pACR6 was more potent than pACR8 in stimulating topoisomerase II mediated DNA cleavage in vitro as well as DNA damage in vivo and pACR6 was accordingly more cytotoxic towards various human and murine cell lines than pACR8. Further, marked cross-resistance to pACR6 was seen among a panel of multidrug-resistant (MDR) cell lines over-expressing the MDR1 (multidrug resistance protein 1) ABC drug transporter, while these cell lines remained sensitive towards pACR8. pACR8 was also capable of circumventing drug resistance among at-MDR (altered topoisomerase II MDR) cell lines not over-expressing drug transporters, while pACR6 was not. Two resistant cell lines, OC-NYH/pACR6 and OC-NYH/pACR8, were developed by exposure of small cell lung cancer (SCLC) OC-NYH cells to gradually increasing concentrations of pACR6 and pACR8, respectively. Here, OC-NYH/pACR6 cells were found to over-express MDR1 and, accordingly, displayed active transport of 3H-labeled vincristine, while OC-NYH/pACR8 cells did not, further suggesting that pACR6, but not pACR8, is a substrate for MDR1. Our results show that the spatial orientation of podophyllotoxin and acridine moieties in hybrid molecules determine target interaction as well as substrate specificity in active drug transport.

Aclarubicin↗

PubMed1998

Inhibition of PNA triplex formation by N4-benzoylated cytosine.

The synthesis of N-((N4-(benzoyl)cytosine-1-yl)acetyl)- N -(2-Boc-aminoethyl)glycine (CBz) and the incorporation of this monomer into PNA oligomers are described. A single CBzresidue within a 10mer homopyrimidine PNA is capable of switching the preferred binding mode from a parallel to an antiparallel orientation when targeting a deoxyribonucleotide sequence at neutral pH. The resulting complex has a thermal stability equal to that of the corresponding PNA-DNA duplex, indicative of a strong destabilization of Hoogsteen strand PNA binding due to steric interference by the benzoyl moieties. Accordingly, incorporation of the CBz residue into linked PNAs (bis-PNAs) results in greatly reduced thermal stability of the formed PNA:DNA complexes. Thus, incorporation of the CBz monomer could eliminate the stability bias of triplex-forming sequences in PNA used in hybridization arrays and combinatorial library formats. Furthermore, it is shown that the benzoyl moiety does not severely interfere with Watson-Crick hydrogen bonding, thereby presenting an interesting route for novel cytosine modifications.

Benzoates↗

PubMed1997

Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine.

The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the T m of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 degrees C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment.

2-Aminopurine↗

PubMed1997

Improvements in automated PNA synthesis using Boc/Z monomers.

An optimized automated PNA synthesis protocol is reported. Under optimal conditions the product yield of a test 17-mer PNA is approximately 90%. The average coupling yield is 99.4%. The synthesis strategy is Boc/Z and the deprotected amine is neutralized in situ. The monomers are added in molar excess to HATU and pre-activated for 60 s before delivery to the resin. The concentration of the activated monomers is 0.08 M during the couplings. Heteroselective solvation provides the highest coupling yields. Acetic anhydride is used as capping reagent followed by a piperidine wash. The protocol has been developed in a 5 mumol scale but is easily scaled up to 10-50 mumol scale syntheses on the automated synthesizer (ABI 433A).

Biopolymers↗

PubMed1996

Effects of pancreatic spasmolytic Polypeptide (PSP) on epithelial cell function.

Trefoil peptides are expressed near endodermal ulcerations and may modulate epithelial repair. The trefoil pancreatic spasmolytic polypeptide (PSP) was tested for growth activity in vitro on epithelial cells and in vivo following intragastric or intravenous infusion in parenterally fed intact rats. Ion transport was assessed as changes in short-circuit current in rat intestine and adenocarcinoma cells in Ussing chambers. PSP stimulated growth of MCF-7 and Colo-357 cells, but only in the presence of extracellular glutathione (GSH). The effect was attenuated by GSH depletion with buthionine sulphoximine, even in GSH-containing media. When GSH-reduced PSP was carboxymethylated with iodoacetic acid, it still depended on extracellular GSH for its growth effect. Intestinal epithelial proliferation in rats was not affected by either intravenous or intraluminal infusion. PSP had no effect on basal or stimulated ion flux in rat jejunum or epithelial monolayers. The peptide did not compete with 125I-labeled epidermal growth factor for its receptor. [14C]Iodoacetamide treatment of PSP, followed by prolonged tryptic digestion yielded predominantly a 14C-labeled tetrapeptide fragment containing Cys1O4, with a lesser quantity of a 14C-labeled 15-amino-acid peptide containing Cys95 (molar ratio 15:1). GSH may predominantly reduce the Cys6-Cys1O4 terminal disulphide bond in PSP. We conclude that some epithelia may exhibit a growth response to PSP if extracellular GSH is present. Reduction of PSP by GSH is not necessary for this response, suggesting that the trefoil receptor or its signal transduction is GSH sensitive. PSP could assist wound healing by interactions with epithelial cells exposed concurrently to a local high GSH concentration.

Animals↗

PubMed1995

Osmo-expression and fast two-step purification of Escherichia coli translation termination factor RF-3.

The gene for the translation termination factor RF-3 in Escherichia coli has recently been cloned and sequenced. Only small amounts of the protein have been purified until now, not sufficient for detailed investigation of the structure and function of this factor. For such studies, we have developed an overexpression system and a purification procedure suitable for large quantities of RF-3. The gene prfC was cloned into the osmo-inducible plasmid pOSEX3 and subsequently transformed into the E. coli strain MKH13. The expression of prfC in this plasmid, which is under the control of the osmotic pressure in the growth medium, leads to a level of RF-3 more than 100-times higher than that in wild-type cells. Using a new two-step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Sepharose HP, we obtain 220 mg pure RF-3 from 10 g overproducing cells, corresponding to 55 mg RF-3/l medium. The identity of the purified protein was confirmed by matrix-assisted laser desorption/ionisation mass spectrometry of tryptolytic fragments and by N-terminal amino acid sequencing. The activity of the purified factor was tested in vitro by measuring the stimulation of RF-2 dependent formylmethionine release from a ribosomal termination complex and the binding capacity of GTP and GDP. All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg.

Amino Acid Sequence↗

PubMed1995

Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice.

BACKGROUND/AIMS: Epidermal growth factor (EGF) is present in gastric juice and has potent mitogenic properties. The stability of EGF in gastric juice under various physiological and pathophysiological conditions was examined. METHODS: Recombinant human EGF1-53 was incubated with HCl containing pepsin. We also determined the forms of EGF present in the gastric juice of patients under basal conditions, patients taking the acid suppressant omeprazole, patients with achlorhydria, and volunteers undergoing intragastric neutralization with NaHCO3 (n = 6 per group). Samples were analyzed using mass spectroscopy and/or high-pressure liquid chromatography followed by radioimmunoassay. The effect of acid and pepsin digestion on EGF bioactivity was determined using an in vitro hepatocyte bioassay and an in vivo cytoprotection assay in the rat stomach. RESULTS: EGF1-53 was digested to the EGF1-49 and EGF1-46 forms in all samples containing pepsin when the pH was < 4. In gastric juice samples with pH > 4, the proportion of intact EGF increased to about 60%. For both methods of bioassay, intact EGF1-53 was about 3-4 times as potent as acid and pepsin-treated EGF. CONCLUSIONS: EGF is produced in the 1-53 form but is rapidly cleaved to smaller, less active forms in acidic gastric juice. In contrast, only a small proportion of the EGF is cleaved if the pH is maintained above 4. This mechanism may be relevant to the healing process of acid suppressants.

Achlorhydria↗

PubMed1994

Peptide ladder sequencing by mass spectrometry using a novel, volatile degradation reagent.

A conceptually novel approach to protein sequencing involves the generation of ragged-end polypeptide chains followed by mass spectroscopic analysis of the resulting nested set of fragments. We report here on the synthesis and development of a volatile isothiocyanate (trifluoroethylisothiocyanate) that allows the identification of several consecutive residues starting with a few picomoles of peptide. The nested set of peptides is generated simply by adding equal aliquots of starting peptide each cycle and driving both the coupling and cleavage reactions to completion. No additional reagents are required to act as chain terminators and retention of the peptide terminal amine allows for subsequent modification with quaternary ammonium alkyl NHS esters to improve sensitivity. Complex washing procedures are not required each cycle, as reagents and by-products are efficiently removed under vacuum, eliminating extractive loss. Multiple peptide samples can be processed simultaneously, with each degradation cycle completed in 35-40 min. The inherent simplicity of the process should allow for easy automation and permit rapid processing of samples in parallel.

Amino Acid Sequence↗

PubMed1993

New compounds related to podophyllotoxin and congeners: synthesis, structure elucidation and biological testing.

4-Azido, 4-amino, 4-amido and 4-alkoxy compounds related to the lignans podophyllotoxin and 4'-demethylepipodophyllotoxin have been synthesized, and their structures elucidated. The Ritter reaction was shown to be useful in the preparation of the 4-amido compounds with the required stereochemistry. A preparative method for 4-chloro-4-deoxypicrophyllotoxin, for which all earlier synthetic attempts resulted in the two dehydrated compounds, alpha- and beta-apopicropodophyllotoxin, was developed. Supplementary preliminary studies of the biological activities of some of the compounds were performed. All compounds had pronounced inhibitory effect on the in vitro growth of human cervical cancer cells and TC-mouse cells with 4-amino-4-deoxypodophyllotoxin and 4-azido-4-deoxypodophyllotoxin showing the highest activity. Alkaline elution studies indicate that the toxicity of the 4'-demethoxy derivatives is due to protein-mediated DNA nicking. None of the compounds were found to have antiviral effect against herpes simplex type 2 (HSV-2), human immunodeficiency (HIV), and cytomegalovirus (CMV) in doses not toxic to the cells.

Animals↗

PubMed1991

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum.

A 7.5-kDa protein has been isolated from chlorosomes of Chlorobium limicola f. thiosulfatophilum and the complete primary structure determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The 74-residue protein shows great homology to a similar protein of unknown function which has been isolated from Pelodictyon luteolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the chlorosome is discussed.

Amino Acid Sequence↗

PubMed1990

Localization of epsilon-lysyl-gamma-glutamyl cross-links in five human alpha 2-macroglobulin-proteinase complexes. Nature of the high molecular weight cross-linked products.

Covalent binding of proteinases by human alpha 2-macroglobulin (alpha 2M) results primarily from the formation of stable epsilon-Lys-gamma-Glu isopeptide bonds. Cross-linking engages 12, 13, and 10 of the 14, 14, and 11 Lys residues in chymotrypsin, trypsin, and subtilisin, respectively, and reaction with the alpha-amino group of the C-chain of chymotrypsin and the B-chain of beta-trypsin is also seen. In contrast, cross-linking engages only 6 of the 11 Lys residues in thermolysin. In each of these proteinases, a few residues react to the greatest extent: Lys36, Lys79, Lys87, and Lys93 in chymotrypsin; Lys87, Lys109, Lys222, and Lys239 in trypsin; Lys12, Lys43, and Lys141 in subtilisin; and Lys210 and Lys219 in thermolysin. In elastase, 1 of the 3 Lys residues (Lys87) is tentatively identified as being cross-linked. Formation of unstable bonds judged to be mainly p-tyrosyl-gamma-glutamyl esters can also be significant for some proteinases. In each of the proteinases, several of the strongly reacting Lys residues are located relatively close to each other, presumably reflecting steric constraints within the alpha 2M-proteinase complexes as they form. Proteinases are covalently bound to alpha 2M to one or two of its COOH-terminal bait region-cleaved half-subunits. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of the high molecular weight cross-linked species indicates that binding of a proteinase through two cross-links occurs not only within the 360-kDa disulfide-bridged alpha 2M dimer but also between the two dimers in the alpha 2M tetramer.

Amino Acid Sequence↗

PubMed1989

Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence.

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.

Acyl Coenzyme A↗

PubMed1987

The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65-kilodalton fragment of IF2 from Escherichia coli.

Protein synthesis initiation factor 2 (IF2) is present in Escherichia coli cells as two forms which are expressed from the same gene: IF2 alpha [97.3 kilodaltons (kDa)] and IF2 beta (79.7 kDa). During isolation, a smaller form, IF2 gamma, is generated, presumably by partial proteolysis. It has been purified to homogeneity and has an apparent mass of 70 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectrophoresis of IF2 alpha and IF2 gamma shows that IF2 gamma is immunologically partially identical with IF2 alpha. The sequence of the 15 N-terminal amino acid residues of IF2 gamma was determined and compared with that of IF2 alpha. The N-terminal amino acid of IF2 gamma corresponds to Arg-290 of IF2 alpha, suggesting that IF2 gamma is generated by proteolytic cleavage of the Lys-289-Arg-290 bond of IF2. Assuming a C terminus identical with IF2 alpha, we calculate that IF2 gamma comprises 601 amino acid residues and has a mass of 64.8 kDa. The truncated protein was tested for activities characteristic of IF2 in three in vitro assays: fMet-tRNA(fMet) binding to 70S ribosomes, N-terminal dipeptide synthesis in a DNA-dependent transcription/translation system, and ribosome-dependent GTP hydroly97-7. The specific activities of IF2 gamma were comparable with, or only slightly less than, those for IF2 alpha, indicating that IF2 gamma contains the active centers for interaction with fMet-tRNA(fMet), ribosomes, and GTP. A central region in the primary structure of IF2 shows extensive sequence homology with a number of GDP-binding proteins and especially with the G-domain of elongation factor Tu (EF-Tu).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1986

Characterization of preprocholecystokinin products in the porcine cerebral cortex. Evidence of different processing pathways.

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.

Amino Acid Sequence↗

PubMed1986

Thermal chemistry of podophyllotoxin in ethanol and a comparison of the cytostatic activity of the thermolysis products.

Podophyllotoxin (1) in buffered ethanolic solution is degraded by two pathways. One leads to (a) picropodophyllin (2), which undergoes dehydration to give alpha-apopicropodophyllin (5), which rearranges to give beta-apopicropodophyllin (6), (b) the ethyl ether of picropodophyllotoxin, 8, and (c) the ethyl ether of epipicropodophyllotoxin, 7. The other pathway leads directly to epipodophyllotoxin (10) and the corresponding ethyl ether, 9, and possibly, via a transient 3,4-dehydropodophyllotoxin (5'), to beta-apopicropodophyllin (6). The 1H NMR spectra of these compounds are described, their in vitro cytostatic activity compared, and their syntheses, including that of podophyllotoxin ethyl ether, reported.

Antineoplastic Agents↗

PubMed1986

Extraction and characterization of IgG Fc receptors from group C and group G streptococci.

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.

Amino Acid Sequence↗