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G Petersen

Publications and source records attributed to G Petersen.

At least 37 records · Page 2Linked to original sources

A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to n-acylethanolamine formation in vitro.

Activation of phospholipase D (PLD) is involved in a number of signal transduction pathways in eukaryotic cells. The most common method for determination of PLD activity in vitro involves incubation with a radiolabeled substrate and lipid extraction followed by thin-layer chromatography in order to separate and quantify substrate and product(s). A more rapid assay can be used when utilizing phosphatidylcholine as a substrate because one of the products, choline, is water soluble and therefore easily separated from the substrate. However, this separation principle is not applicable in evaluating N-acylphosphatidylethanolamine (NAPE)-hydrolyzing PLD activity, which produces two lipophilic products, N-acylethanolamine (NAE) and phosphatidic acid. Therefore, we developed a rapid assay for the routine detection of NAPE-hydrolyzing PLD activity. This assay is based on precipitation of radiolabeled substrate (NAPE) in the presence of ZrOCl(2), followed by quantification of radiolabeled NAE released into a methanolic supernatant. The precipitation involves a chemical reaction of the zirconyl cation with the phosphate anion. Conditions were optimized for the complete precipitation of NAPE, whereas N-acyllysophosphatidylethanolamine and glycerophospho(N-acyl)ethanolamine were precipitated at least 95%. Furthermore, this precipitation method can be extended to assays of other anionic phospholipid-hydrolyzing PLD activities by selecting an optimal pH of the precipitation solution. For example, 98;-99% precipitation of phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine was achieved.Consequently, this new assay allows for a convenient examination of PLD activities toward a variety of phospholipid substrates, and in particular allows for the analysis of NAE formation from NAPE in vitro, a feature that will facilitate a more complete biochemical characterization of this anandamide-generating enzyme.

Animals↗

N-acylphosphatidylethanolamine-hydrolysing phospholipase D lacks the ability to transphosphatidylate.

The N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) generates N-acylethanolamines, including N-arachidonoyl-ethanolamine (anandamide), that may be neuroprotective and analgesic. The properties of NAPE-PLD from rat heart and brain microsomes are investigated and compared to those of other PLDs. NAPE-PLD is inhibited by the fatty acid aminohydrolase inhibitor MAFP in high concentrations (> or = 100 microM) while PMSF in high concentrations (10 mM) tends to stabilise NAPE-PLD activity. Oleate inhibits NAPE-PLD but the enzyme is not affected by PIP2, alpha-synuclein or mastoparan. Furthermore, it is for the first time reported that NAPE-PLD is not capable of catalysing a transphosphatidylation reaction like most other known PLDs.

Animals↗

Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).

To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed. Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen. Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p. Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p. The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p. Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction. Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p. The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues. The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine. Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12. The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p. Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes.

Amino Acid Sequence↗

Formation of N-acyl-phosphatidylethanolamine and N-acylethanolamine (including anandamide) during glutamate-induced neurotoxicity.

N-Acyl-phosphatidylethanolamine (NAPE) is present in very small amounts in mammalian tissues (less than 0.1% of total phospholipids). However, NAPE as well as its degradation product, N-acylethanolamine (NAE), can be formed in certain neuronal tissues in response to increased [Ca2+]i. A high [Ca2+]i will activate the NAPE-forming N-acyltransferase using the sn-1 acyl group of a donor phospholipid as substrate in the transfer reaction. This membrane-bound enzyme seems to have no substrate specificity with respect to transfer of acyl groups; thus the fatty acids in the N-acyl group of NAPE are mainly 16:0 and 18:1, corresponding to the fatty acids in the sn-1 acyl group of the donor phospholipids. The NAPE-hydrolyzing phospholipase D also seems not to be acyl-group specific. In mouse neocortical neurons in primary culture, formation of NAPE and NAE is stimulated by glutamate via activation of the N-methyl-D-aspartate-receptor. Both NAPE and, to a lesser extent, NAE accumulate in a linear fashion for many hours while at the same time the neurons are dying. Likewise, in neurons prelabeled with 14C-arachidonic acid, 14C-arachidonic acid-labeled NAPE, and anandamide (= N-arachidonoylethanolamine) are accumulating. The formation of NAPE and NAE may represent a cytoprotective response in relation to various forms of neurotoxicity.

Acyltransferases↗

Percutaneous endoscopic gastrostomy in children with cancer.

We reviewed the clinical course of 32 children with cancer who received nutrition through a feeding tube placed percutaneously during gastroscopy (PEG). Their median age was 5.1 y (75%, range: 1.8-13.7 y, min: 3.5 mo) when the PEG was done 0.7-23 mo after diagnosis (median: 1.8 mo, 75%; range: 0.9-8 mo). Five of the children underwent bone marrow transplantation with the gastrostomy in place. There was a significant (p = 0.0001) decrease in the median weight-for-age SDS of 0.55 (75%, range: -1.18-0.28) from the time of diagnosis to placement of the gastrostomy. Twenty-two percent of the children had neutrophils < 0.5 x 10(9)l at the time of placement. There were no major postoperative complications. Seventy-two percent of the patients experienced a total of 55 minor and transient complications including leakage of gastric juice (n = 29), superficial wound infections (n = 23), mechanical problems (n = 2), or bleeding (n = 1). There were no documented cases of bacteraemia. Twelve of the wound infections (52%) arose during neutropenic episodes. Two tubes were replaced due to mechanical problems. There was a median increase in weight SDS of 0.3 (75%, range: -0.6-1.1) from the time of placing the gastrostomy to the end of follow-up (p = 0.054). Nutrition via gastrostomy in children with cancer has several advantages. It is rarely associated with more than minor complications, it is cosmetically more acceptable than the nasogastric tube and it improves nutrition at far lower cost than parenteral nutrition. In selected cases in which bone marrow transplantation or intensive treatment protocols are planned, we suggest that a gastrostomy should be considered before malnutrition develops.

Adolescent↗

[Effectiveness of vitamin E in comparison with diclofenac sodium in treatment of patients with chronic polyarthritis].

In a randomized, double blind parallel group comparison the antiphlogistic and analgetic efficacy of high-dosed vitamin E (3 x 400 mg RRR-alpha-Tocopherolacetat/d) versus diclofenac-sodium has been investigated in hospitalized patients with established chronic rheumatoid arthritis. After 3 weeks of treatment the vitamin E group (n = 42) as well as the diclofenac group (n = 43) showed a significant improvement of all assessed clinical parameters. Duration of morning stiffness could be reduced under vitamin E treatment from 90 min to 68 min and under diclofenac treatment from 68 min to 30 min. The joint index according to Richie declined from 56 to 46 (vitamin E) and 49 to 34 (diclofenac). Grip strength increased in the vitamin E group as well as in the diclofenac group. In addition, the degree of pain, assessed by a 10 cm visual analogue scale, reduced significantly under vitamin E as well as under diclofenac. Regarding the therapeutical result both, physicians and patients, considered both drugs to be similarly effective. Especially regarding the risk profile of NSAR in long-term treatment of chronic rheumatoid arthritis intake of high-dosed vitamin E is a possible alternative in the treatment of inflammatory rheumatoid diseases.

Adult↗

Bioavailability of escin after administration of two oral formulations containing aesculus extract.

In a steady-state cross-over study in 18 healthy volunteers, the relative bioavailability of beta-escin (CAS 11072-93-8) after oral administration of a new immediate release enteric-coated test formulation containing aesculus extract was evaluated in comparison with a prolonged-release reference preparation. The subject received the test and the reference preparation in randomised sequence for 7 days each with no washout period in between. The daily dose was 50 mg escin b.i.d. Blood samples for pharmacokinetic profiling were taken on the 7th treatment day of each period over a full 24-h cycle of two successive dosing intervals. For the determination of beta-escin serum concentrations, a highly specific radioimmunoassay (RIA) was used. Generally, escin serum concentrations were lower during the second dosing interval (night) than during the first interval, probably indicating a drug by food interaction. (The morning dose was given after overnight fasting whereas the evening dose was given between meals). Test and reference demonstrated bioequivalence with regard to the extent of absorption; for the AUC (0-24 h p.a.), the 90% confidence interval ranged from 84% to 114% (point estimate: 98%). The differences observed for rate parameters can be disregarded due to the generally slow elimination and the wide therapeutic concentration range of escin.

Administration, Oral↗

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

Amino Acid Sequence↗

Recommendations for follow-up care of individuals with an inherited predisposition to cancer. II. BRCA1 and BRCA2. Cancer Genetics Studies Consortium.

OBJECTIVE: To provide recommendations for cancer surveillance and risk reduction for individuals carrying mutations in the BRCA1 or BRCA2 genes. PARTICIPANTS: A task force with expertise in medical genetics, oncology, primary care, gastroenterology, and epidemiology convened by the Cancer Genetics Studies Consortium (CGSC), organized by National Human Genome Research Institute (previously the National Center for Human Genome Research). EVIDENCE: Studies evaluating cancer risk, surveillance, and risk reduction in individuals genetically susceptible to breast and ovarian cancer were identified using MEDLINE (National Library of Medicine) and from bibliographies of articles thus identified. Indexing terms used were "genetics" in combination with "breast cancer," "ovarian cancer," and "screening," or "surveillance" in combination with "cancer family" and "BRCA1" and "BRCA2." For studies evaluating specific interventions, quality of evidence was assessed using criteria of the US Preventive Services Task Force. CONSENSUS PROCESS: The task force developed recommendations through discussions over a 14-month period. CONCLUSIONS: Efficacy of cancer surveillance or other measures to reduce risk in individuals who carry cancer-predisposing mutations is unknown. Based on expert opinion concerning presumptive benefit, early breast cancer and ovarian cancer screening are recommended for individuals with BRCA1 mutations and early breast cancer screening for those with BRCA2 mutations. No recommendation is made for or against prophylactic surgery (eg, mastectomy, oophorectomy); these surgeries are an option for mutation carriers, but evidence of benefit is lacking, and case reports have documented the occurrence of cancer following prophylactic surgery. It is recommended that individuals considering genetic testing be counseled regarding the unknown efficacy of measures to reduce risk and that care for individuals with cancer-predisposing mutations be provided whenever possible within the context of research protocols designed to evaluate clinical outcomes.

Antineoplastic Agents, Hormonal↗

Recommendations for follow-up care of individuals with an inherited predisposition to cancer. I. Hereditary nonpolyposis colon cancer. Cancer Genetics Studies Consortium.

OBJECTIVE: To provide recommendations for cancer surveillance and risk reduction for individuals carrying mutations associated with hereditary nonpolyposis colon cancer (HNPCC). PARTICIPANTS: A task force with expertise in medical genetics, oncology, primary care, gastroenterology, and epidemiology convened by the Cancer Genetics Studies Consortium (CGSC), organized by the National Human Genome Research Institute (previously the National Center for Human Genome Research). EVIDENCE: Studies evaluating cancer risk, surveillance, and risk reduction in individuals genetically susceptible to colon cancer were identified using MEDLINE and bibliographies of articles thus identified. Indexing terms used were "genetics" in combination with "colon cancer," and "screening" in combination with "cancer family" and "HNPCC." For studies evaluating specific interventions, quality of evidence was assessed using criteria of the US Preventive Services Task Force. CONSENSUS PROCESS: The task force developed recommendations through discussions over a 14-month period. CONCLUSIONS: Efficacy of cancer surveillance or other measures to reduce risk in individuals who carry cancer-predisposing mutations is unknown. Based on observational studies, colonoscopy every 1 to 3 years starting at age 25 years is recommended for individuals known to have HNPCC-associated mutations. Endometrial cancer screening is also recommended, based on expert opinion concerning presumptive benefit. No recommendation is made for or against prophylactic surgery (ie, colectomy, hysterectomy); these surgeries are an option for mutation carriers, but evidence of benefit is lacking. It is recommended that individuals considering genetic testing be counseled regarding the unknown efficacy of measures to reduce risk and that care for individuals with cancer-predisposing mutations be provided whenever possible within the context of research protocols designed to evaluate clinical outcomes.

Adaptor Proteins, Signal Transducing↗

Transcription of DmRP140, the gene coding for the second-largest subunit of RNA polymerase II.

To analyze transcriptional control regions of Drosophila melanogaster housekeeping genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Upstream of DmRP140 the genomic region harbors a gene which is transcribed in the opposite direction (DmRP140up). By determination of the transcription start sites of both genes we found a short non-transcribed intergenic region of 220 bp. Functional analysis of various promoter reportergene constructs by transient transfection of cultured cells revealed that sequences important for transcription of DmRP140 are located in the untranslated leader of the upstream gene. The onset of DmRP140 transcription during embryonic development was studied in transgenic flies using beta-galactosidase as reportergene. To distinguish between the maternally provided DmRP140 transcripts and the embryonically transcribed RNA the offspring of nontransformed females and male transformants was examined. The development of a sensitive detection assay based on a chemiluminescent substrate for beta-galactosidase allowed us to determine the onset of DmRP140 transcription to between 8-10 h after oviposition. Thus, DmRP140 transcription does not start following the transcriptional transition period between 2-3 h of development but occurs much later in embryogenesis coinciding with decreasing DNA synthesis and cell division rates.

Animals↗

Identification of the gene coding for the largest subunit of RNA polymerase I (A) of Drosophila melanogaster.

The gene coding for the largest subunit (RPA1) of RNA polymerase I (A) of Drosophila melanogaster (DmRPA1) was cloned and sequenced. The gene is interrupted by seven small introns and the cDNA reveals an open reading frame of 4932 nucleotides. The deduced polypeptide consists of 1644 amino acids with a calculated molecular weight of 185 kDa. Although the protein sequence exhibits the specific pattern of conserved regions found in all RNA polymerase largest subunits characterized so far, the overall sequence similarity among the RPA1 subunits of different species is much lower than seen with the corresponding subunits of RNA polymerases II and III. Two highly divergent hydrophilic domains characteristic for RPA1 separate the conserved blocks a and b in the N-terminal region and blocks g and h in the C-terminal section, respectively. In both cases the distance between the homologous blocks is enlarged by about 70 amino acids relative to the largest subunits of RNA polymerases II and III, and the corresponding subunit of the archaebacterial enzyme. Compared with RPA1 sequences of lower eukaryotes, the C-terminal hydrophilic domain in DmRPA1 is similar in length and acidity whereas the N-terminal domain is slightly shorter but retains the same basicity. The sequence insertions do not feature common motifs, suggesting a role for them in the interaction of RNA polymerase I with proteins required for the species-specific transcription of rDNA. The RPA1 subunits of Drosophila melanogaster and lower eukaryotes share an additional Zn-binding motif at the N-terminus with archaebacterial and RPC1 subunits, testifying to the complex evolutionary relationships among the RNA polymerases.

Amino Acid Sequence↗

Phylogenetic analysis of the Triticeae (Poaceae) based on rpoA sequence data.

A phylogenetic analysis was conducted on 31 diploid species representing 21 of the 24 monogenomic genera of the Triticeae. The data used were derived from a 1343- to 1358-bp region of the plastid genome spanning the entire rpoA gene plus minor parts of the petD and rps11 genes and the two intergenic spacers surrounding rpoA. Bromus inermis (Bromeae) was used as an outgroup. A total of 68 variable sites, 25 of them phylogenetically informative, and seven length mutations were detected. The length mutations occurred in the noncoding regions. Phylogenetic analyses were performed on the whole data set and on various subsets. The analysis of the unweighted data resulted in 48 equally parsimonious trees (length 98, CI = 0.88, RI = 0.92, ti = 0.25). A parsimony jackknife analysis proved several clades to be well supported. The effect of transition/transversion weighting was also investigated. In general, congruence with other data sets was negatively effected by weighting. The preferred phylogenetic hypothesis is congruent with a phylogeny based on plastid RFLP data including the same taxa, but it is largely incongruent with phylogenies derived from nuclear rDNA and morphology.

Base Sequence↗

Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies.

Amino Acid Sequence↗

PCR and sequencing from a single pollen grain.

In order to eliminate the laborious step of DNA extraction preceding all studies within the field of plant molecular biology we attempted to do PCR amplifications directly on pollen grains. Successful PCR amplification was obtained in reactions including a single pollen grain from Hordeum vulgare or Secale strictum. Both the plastid gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) and the nuclear-encoded internal transcribed spacer regions (ITS) and the 5.8S rDNA region were amplified and sequenced to verify PCR amplification.

Hordeum↗

The effects of a new low-dose combined oral contraceptive containing levonorgestrel on ovarian activity.

OBJECTIVE: The study was designed to evaluate the effects of a new low-dose monophasic combined oral contraceptive (100 micrograms levonorgestrel/20 micrograms ethinylestradiol) on ovarian activity. METHOD: This was an open-label study in 15 healthy female volunteers. After an ovulatory pretreatment cycle, subjects were treated for three consecutive cycles. RESULTS: Out of 15 healthy females with an ovulatory pretreatment cycle, 13 completed the study and two dropped out. In ten females, various degrees of ovarian activity were characterized, mainly in the second and third treatment cycle. In eight cases follicle-like structures either persisted or developed throughout the pill-free interval, growing up to luteinized unruptured follicles or cyst-like structures that reduced their size or disappeared in the following treatment cycle. CONCLUSION: Ovarian follicular activity was significantly suppressed during treatment with levonorgestrel 100 micrograms/ethinylestradiol 20 micrograms. Similarly, endogenous ovarian steroid production was almost uniformly decreased. A 'dissociation' of ovarian and endometrial development was also seen during this therapy.

Adolescent↗

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries.

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50-200 bp in length and cloning these into the 5' terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

Amino Acid Sequence↗

[Steady homosexual relationship and HIV infection].

In order to study the relative impact of a steady homosexual relationship on the presence of HIV antibodies, a cross-sectional study was undertaken of 719 homosexual men in Oslo during the period of 1983-1987. 74/719 (10%) were HIV positive. 37/74 (50%) of the HIV positives and 185/645 (29%) of the HIV negatives were having a steady homosexual relationship that had lasted six months or more (chi-square test, p < 0.05). In a multivariate logistic regression analysis the adjusted odds ratio of being HIV positive was 2.3 (1.3-4.3, 95% confidence interval (CI)) for homosexual relationship lasting 1/2-4 years and 3.1 (1.5-6.6, 95% CI) for homosexual relationship lasting 5 years or more as compared with not being in a homosexual relationship. The number of male partners and frequent anal intercourse were also significantly associated with presence of HIV antibodies. The results suggest that situational variables associated with a steady partnership may be barriers to HIV-preventive measures.

Adult↗