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B Kemper

Publications and source records attributed to B Kemper.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Animals↗
PubMed1998

Binding of endonuclease VII to cruciform DNA. Visualization in the electron microscope.

The binding of Holliday structure resolving endonuclease VII to cruciform DNA was studied in the electron microscope. The protein was found to bind either to the junction or to one of the arms or an end of one of the arms of the construct. The amount of bound protein was determined by measuring the size of the complexes. On average, one complex containing three dimers was found per one molecule of cruciform DNA.

Binding Sites↗
PubMed1998

An apyrimidinic site kinks DNA and triggers incision by endonuclease VII of phage T4.

Apurinic/apyrimidinic lesions (AP-sites) occur frequently in DNA, generated by physically and chemically induced or spontaneous loss of bases. Repair mechanisms have evolved in organisms to deal efficiently with AP-sites by first incising the DNA at the lesion, followed by excision and resynthesis of the damaged strand. Here we report that endonuclease VII (endo VII) of phage T4, which was originally classified as a debranching and Holliday structure resolving enzyme, also recognizes AP-sites with high efficiency. The enzyme cleaves both strands of double-stranded DNA in a stepwise fashion a few nucleotides 3' of the lesion. In a search for a recognition signal shared by all known endo VII substrates, kinking of DNA has earlier been suggested as such a signal. In support of this hypothesis, we demonstrate here that AP-sites induce distinct kinks in synthetic oligonucleotides allowing efficient intramolecular ring closure by ligation.

Bacteriophage T4↗
PubMed1998

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure.

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Base Composition↗
PubMed1998

Endonuclease VII has two DNA-binding sites each composed from one N- and one C-terminus provided by different subunits of the protein dimer.

Endonuclease VII (endo VII) is a Holliday structure-resolving enzyme of bacteriophage T4. Its activity depends on dimerization, DNA binding and hydrolysis of two phosphodiester bonds flanking the Holliday junction. We analysed the DNA-binding activity of truncated monomeric and covalently linked dimeric endo VII proteins. We show that both ends of endo VII are involved in DNA binding. In particular, the C-terminus of one subunit interacts with the N-terminus of the other subunit, constituting one DNA-binding site; the other two termini form the second binding site of the dimer. One binding site is sufficient to bind cruciform DNA. The concerted mechanism involving termini from different subunits ensures that only dimers bind to Holliday structures, thus providing two catalytic centres which introduce two cleavages in opposite strands. This is a precondition for precise resolution of Holliday structures.

Bacteriophage T4↗
PubMed1998

Localization and characterization of the dimerization domain of holliday structure resolving endonuclease VII of phage T4.

Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4. Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA. In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated. Some of these mutations affected the dimerization of Endo VII. Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state. Only the dimeric fraction of this protein bound to DNA. The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde. The dimerization domain was thus determined to reside between amino acid residues 55 and 105. Computer analyses predicted two alpha-helices (H2 and H3) in this section of the protein. As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization. Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them.

Amino Acid Sequence↗
PubMed1998

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding.

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.

Amino Acid Sequence↗
PubMed1998

A conserved proline-rich sequence between the N-terminal signal-anchor and catalytic domains is required for assembly of functional cytochrome P450 2C2.

In cytochrome P450 2C2, the region which links the N-terminal signal anchor with the catalytic domain contains a highly conserved proline-rich region with the sequence, 30-PPGPTPFP-37. Mutation of proline-30 or proline-33 diminished activities of the mutants expressed in COS-1 cells (Chen, C., and Kemper, B. (1996) J. Biol. Chem. 271, 28697-28611). Substitution of alanine, proline, or arginine for glycine-32 abolished laurate hydroxylase activity of the proteins expressed in COS-1 cells, which suggests that this residue is also functionally important. To determine the basis for the decreased activity in COS-1 cells, the activities and spectral properties of mutant proteins expressed in insect cells and bacteria were determined. Substitution of alanine for either proline-30 or -33 resulted in reduced expression in insect cells of functional cytochrome P450 hemoprotein and an increase in the expression of inactive cytochrome P420. In contrast, substitution of alanine for proline-31, -35, or -37 resulted in hemoproteins with spectra similar to cytochrome P450 2C2 so that the amount of cytochrome P450 expressed in insect cells correlated with the activities of the mutants in COS-1 cells. The laurate hydroxylase activities per nanomole of cytochrome P450 in insect microsomes were similar for wild type and all mutants, indicating that, once folded, the catalytic activity of membrane-bound cytochrome P450 was not affected by the mutations. Expression in bacteria resulted in diminished expression of cytochrome P450 for all mutants, with the greatest decrease for the proline-30 and -33 mutants, and increased cytochrome P420. In contrast to the insect cell studies, the proline-30 and -33 mutants were inactive, while the other mutants had specific activities 30-70% of cytochrome P450 2C2. These data are consistent with a role for the proline-rich region in efficient assembly of cytochrome P450 2C2 in eukaryotic cells. Mutations of this region also may affect the conformational integrity of the proteins, which was revealed by assays of solubilized bacterially expressed proteins.

Animals↗
PubMed1998

Enzymatic mutation detection. Procedure for screening and mapping of mutations by immobilised endonuclease VII.

Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.

DNA↗
PubMed1998

Torsional control of double-stranded DNA branch migration.

DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position.

Base Sequence↗
PubMed1998

Repeated sleep deprivation once versus twice a week in combination with amitriptyline.

OBJECTIVE: Although a combination of antidepressive pharmacotherapy with repeated sleep deprivation therapy has proved to be an effective and easily applied treatment strategy, no systematic study investigating the most favourable interval between the sleep deprivation trials is available. METHOD: In a cross-over design, 44 patients with a major depressive episode (ICD-10) were randomly allocated to 4 weeks treatment with amitriptyline (150 mg/d) in combination with either late sleep deprivation (of the second half of the night, LSD) twice weekly during week 1 and 2 followed by LSDs once weekly during week 3 and 4 or vice versa. Two hypotheses were tested: Does LSD twice weekly during week 1 and 2 accelerate the clinical response compared with LSD once weekly? Does LSD twice weekly during week 3 and 4 improve the response rate compared with LSD once weekly? RESULTS: According to both observer rating (Hamilton Rating for Depression, 21-item version) and self-rating (Visual Analogue Mood Scale), no significant differences could be ascertained between the groups compared at any time of the investigation. With respect to drop-outs and immediate LSD effects there is a trend for patients undergoing twice weekly LSDs followed by once weekly LSD trials to have a more favourable treatment course than the control group. CONCLUSION: The hypotheses tested were answered in the negative. "High dose" LSD administration does not prove to be clearly superior to "low dose" LSD. This finding is in line with most of the corresponding data on antidepressive pharmacotherapy.

Adult↗
PubMed1998

Enzymatic mutation detection. Phosphate ions increase incision efficiency of endonuclease VII at a variety of damage sites in DNA.

The ability of endonuclease VII (endo VII) to cleave at mispairings in double-stranded DNA has recently been used for enzymatic mutation detection (EMD) [R. Youil, B.W. Kemper, R.G.H. Cotton, Proc. Natl. Acad. Sci. USA 92 (1995) 87-91]. The method is based on mapping cleavages in heteroduplex DNAs obtained from mutant and wildtype sequences. Despite the capability of endo VII to cleave at all possible mispairings, relative cleavage efficiencies vary considerably for individual mismatches and may escape detection if located in an unfavorable sequence surrounding. We report here improved reaction conditions which can increase the selectivity of the enzyme for mismatches up to 500-fold, as demonstrated with a mutation in a 247 nt long fragment from exon 7 of human gene p53. The new conditions involve replacement of Tris/HCl buffer by phosphate buffer and change from pH 8.0 to 6.5. Various concentrations of phosphate ions should be tried in the assay to meet individual requirements of the substrate.

Base Composition↗
PubMed1998

[Juvenile neuronal ceroid lipofuscinosis (Spielmeyer-Vogt disease)].

BACKGROUND: The neuronal ceroid-lipofuscinosis (NCL) belongs to progressive neurodegenerative disorders of childhood with both ophthalmologic and neurologic symptoms. In the most common type in Germany, the juvenile type, the ophthalmological examination is essential for an early diagnosis. PATIENT: A 5-year-old boy had exhibited a loss of visual acuity, visual field and colour perception in his pre-school age. His clinical features and electrophysiologic data are presented. The final diagnostic clues were drawn from the neuropediatric and cytologic examinations. RESULTS: This patient shows the typical clinical feature of the juvenile NCL with a relatively rapid visual loss with bull's eye maculopathy in the pre-school- to early school age. His electroretinogram was abolished, and his EEG showed pathologic results. The diagnosis could be established by electron microscopy of his lymphocytes. Epilepsy and intellectual defects are expected to set in only few years later. CONCLUSIONS: Although there is no therapy, the ophthalmologist should be aware of this rare entity because prognostic counselling, social help and perhaps genetic counselling could be offered to the families. In many cases, a prenatal diagnosis is possible.

Child↗
PubMed1998

Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2.

Although the induction of drug-metabolizing systems by phenobarbital has been recognized for about 40 years, the mechanism by which cytochrome P450 gene expression is increased is still not well understood. A 163-bp fragment at about -2.2 Kb in CYP2B2 has been shown to mediate phenobarbital induction in primary rat hepatocytes (Trottier, et al. [1995] Gene 158:263-268) and by an in situ transient transfection assay in rat liver (Park, Y., et al. [1996]. J. Biol. Chem. 271:23725-23728). Deletion mutations of this fragment indicated that the 88-bp stretch from -2258 to -2170 was the minimal sequence that could mediate phenobarbital induction in the in situ system if single copies of the deleted fragments fused to the CYP2C1 proximal promoter were assayed. If three copies of the fragments were present, 5' and 3' deletions defined a minimal 37-bp core fragment, which, although necessary for phenobarbital responsiveness, was not sufficient unless additional sequence was present at either end, suggesting that redundant elements were present in the two flanking regions. Site-specific mutagenesis of an NF-1 site within the 88-bp fragment and linker scanning mutagenesis across the fragment indicated that the NF-1 site and a region to the 5' side of the site contributed to the magnitude of the response, but neither the NF-1 mutations nor any of the linker scanning mutations eliminated the response to phenobarbital. Mutation in a region 3' of the NF-1 site resulted in elevated basal expression without substantial effects on phenobarbital-induced expression. Binding of NF-1 to the 37-bp core fragment was established by gel-shift competition studies and by supershifts of the protein-DNA complexes by antisera to NF-1. Additional protein-DNA complexes were detected in the regions flanking the NF-1 site. These studies indicate that the CYP2B2 phenobarbital-responsive enhancer contains multiple constitutive and phenobarbital-responsive elements. Binding of nuclear proteins from control or phenobarbital-treated animals in vitro to this region was very similar. The only difference detected was a complex that was substantially reduced by phenobarbital treatment and mapped to the 3' side of the NF-1 site.

Animals↗
PubMed1998

Regulation of cytochrome P450 gene transcription by phenobarbital.

The ability of phenobarbital to induce levels of drug metabolism in mammals has been known for over 40 years. However, the molecular mechanisms underlying increased expression of the genes of the key enzyme in drug metabolism, cytochrome P450, have not been elucidated, primarily because in vitro model systems in which the induction could be studied were not available. Transfected primary cultured hepatocytes, transfection of liver in situ, and transgenic mice now provide suitable models for phenobarbital induction. In this review, progress toward understanding the mechanism of phenobarbital induction of gene expression is discussed with an emphasis on the mammalian genes, CYP2B1, CYP2B2, and Cyp2b10, which are most highly inducible by phenobarbital. Barbiturate induction of P450s in Bacillus megaterium, which is the system best understood, and its relevance to mammalian mechanisms of induction are also discussed. In B. megaterium, the binding of a repressor to several motifs is reversed by direct effects of barbiturates and by induction of positively acting factors. One of the repressor binding sites, the barbie box, is present in many mammalian phenobarbital-inducible genes, including the promimal promoter regions of CYP2B1, CYP2B2, and Cyp2B10. In the mammalian P450 genes, evidence has been proposed for phenobarbital-regulated elements both in the proximal promoter region and in a distal enhancer region. The role of the proximal region is controversial. A positively acting element that overlaps the barbie box sequence and a negative element have been proposed to mediate induction of CYP2B1/2, based primarily on protein binding and cell-free transcription assays. In contrast, other investigators have not found differences in phenobarbital-dependent protein binding in the proximal promoter region nor mediation of phenobarbital induction by this region. A distal gene fragment, at about -2000 kb in CYP2B1, CYP2B2, and Cyp2b10, has been shown to be a phenobarbital-responsive enhancer independent of proximal promoter elements. This fragment contains several binding sites for proteins and several functional elements, including an NF-1 site, and, therefore, has been designated as a phenobarbital-responsive unit. Possible models are presented in which phenobarbital treatment induces altered chromatin structure, which allows the binding of positively acting factors, or activates factors already bound, to the distal enhancer and the proximal promoter.

Animals↗
PubMed1997

Phenobarbital alters protein binding to the CYP2B1/2 phenobarbital-responsive unit in native chromatin.

Phenobarbital is a classical inducer of the drug metabolizing cytochrome P450 genes, but the molecular mechanism of induction has not been elucidated. Functional analyses have identified a phenobarbital-responsive unit in the rat CYP2B1/2 and mouse Cyp2b10 genes about -2.3 kilobase pairs from the transcriptional start site, but little or no changes in protein binding to this region were observed in vitro. To examine the role of chromatin structure, protein binding to the phenobarbital-responsive unit assessed by in vitro DNase I footprinting was compared with that assessed by DNase I in vivo footprints in native chromatin. A region centered on a putative nuclear factor-1 site was the major protected region in in vitro footprints, and there were no detectable differences in binding between extracts from control and phenobarbital-treated animals. In contrast, phenobarbital treatment dramatically altered the protection pattern in native chromatin. In control samples a core region of about 25 base pairs (bp) centered on the nuclear factor-1 site was protected. However, after phenobarbital treatment, the protection of this core region was increased, and more dramatically the region of protection was extended 20 bp to either side so that a total of about 60 bp were protected. These results provide the first evidence that phenobarbital treatment alters the composition or architecture of proteins binding to the phenobarbital-responsive unit region and indicate that chromatin structure is important in this process. Because proteins are bound to the region in the untreated animal, the mechanism of induction involves the activation of proteins bound to the region and possibly recruitment of additional regulatory proteins rather than conversion of a closed chromatin structure to an open one that can bind regulatory factors.

Animals↗