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P Krieg

Publications and source records attributed to P Krieg.

At least 37 records · Page 2Linked to original sources
PubMed1997

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform.

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis-type 12-lipoxygenase (e12-lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4-8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)-HETE from arachidonic acid. By fluorescence in situ hybridization the e12-lipoxygenase gene was localized to chromosome band 11 B1-B3.

Amino Acid Sequence↗
PubMed1997

Xbap, a vertebrate gene related to bagpipe, is expressed in developing craniofacial structures and in anterior gut muscle.

The Drosophila bagpipe (bap) gene is involved in the specification of the musculature of the embryonic midgut. We report the isolation and characterization of a Xenopus sequence, Xbap, which is closely related to bap. Xbap is also expressed in the developing musculature of the midgut, suggesting that this developmental role of bagpipe is evolutionarily conserved. However, a second, novel role in development is suggested by the observation that Xbap is also expressed in a region of the developing facial cartilage. Using a combination of cartilage staining and comparison to the goosecoid head expression pattern, we show that Xbap expression marks the precursors to the basihyobranchial, palatoquadrate, and possibly Meckel's cartilages. This vertebrate bagpipe sequence therefore is expressed in both mesodermally and neural crest-derived tissues.

Amino Acid Sequence↗
PubMed1996

Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells.

The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies. Purified KLBP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2). Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent Kd of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K(d) of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all. The protein did not bind prostaglandin E2 with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K(d) of 848 +/- 211 nM) and 15-HPETE (Kd of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (Kd of 1560 +/- 115 nM and greater than 4 microM, respectively). all-trans-Retinoic acid was a weak ligand for KLBP, binding with a Kd of 3600 nM, and all-trans-retinol did not displace 1,8-ANS. Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that the side chains of one or more cysteine residues may reside within the putative ligand-binding cavity. Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid. These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.

Amino Acid Sequence↗
PubMed1996

Determination of alpha-amylase activity using Fourier transform infrared spectroscopy.

A new method for the determination of alpha-amylase activity in aqueous solutions and human serum with FTIR-spectroscopy is proposed. The chemical reaction catalyzed by the enzyme under study can be followed directly when applying FTIR-spectroscopic detection also in the case, where no colored or electrochemical active species are generated or consumed during the course of the reaction of alpha-amylase with simple starch. Therefore the determination of the alpha-amylase activity could successfully be performed by recording two FTIR-spectra, one immediately after mixing the sample and a substrate (starch-) solution and the other after a 20 min reaction time. From these two FTIR-spectra a difference spectrum was calculated hereby eliminating an unspecific absorption of the matrix. The intensities of the resulting difference spectra corresponded to the extent of the reaction which took place during the investigated time interval and hence could be related to the activity of the enzyme in the sample. The developed method is linear from 80 to 1400 U/l (r.s.d.=5% for 700 U/l) in aqueous solutions and was also successfully applied to the determination of alpha-amylase activity in human serum where a linear working range from 100 to 800 U/l (r.s.d.=11% for 150 U/l) was achieved.

Journal Article↗
PubMed1995

12-Lipoxygenase isoenzymes in mouse skin tumor development.

12-lipoxygenase-catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12-lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50- to 60-fold more 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell-free preparations of the tumors exhibited about fivefold elevated 12-lipoxygenase activities. A papilloma-derived cDNA library was screened with human platelet-type 12-lipoxygenase cDNA probes. Two cDNA clones encoding the platelet-type and the leukocyte-type isoforms of murine 12-lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS-7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte-type enzyme metabolizing linoleic and arachidonic acid to 13-hydroxy-9,11-octadecadienoic acid and to 12- and 15-HETE, respectively, and the platelet-type enzyme oxygenating exclusively arachidonic acid to 12-HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet-type 12-lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet-type mRNA, whereas leukocyte-type specific transcripts were detectable only in a few tumors. These data suggest that the platelet-type enzyme is the 12-lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12-HETE found in neoplastic epidermis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1993

Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterization of a cloned sequence activated during multistage carcinogenesis in mouse skin.

Differential screening of cDNA libraries from chemically induced malignant mouse skin squamous cell carcinomas (SCCs) identified sequences, including one called mal1, that were up-regulated in their expression at both the benign papilloma and the malignant SCC stages during tumor development. The mal1 plasmid cDNA clone was used to screen lambda phage cDNA libraries made from chemically induced papillomas and SCCs. Two size classes (655 and 933 nucleotides excluding the poly(A) tail) of full-length cDNAs were isolated. The corresponding mRNAs differ in their 3'-untranslated region by 278 nucleotides as a result of utilizing two alternative polyadenylation signals. Both transcripts were expressed simultaneously, showing the same expression patterns, with the smaller one being the predominant species. Most tissues examined showed a weak expression of mal1 mRNA. High levels of mal1 transcripts could be detected in adipose and mammary tissues and tongue epithelia and predominantly in epidermis. The expression observed in epidermis was up-regulated dramatically during tumor formation. Computer-assisted sequence analysis revealed one open reading frame that encoded a protein of 135 amino acid residues with extensive homology to members of the lipid-binding protein family. Residues determining the proposed beta-clam structure of these proteins and the structure of the lipid-binding region were shown to be conserved in the mal1 gene. In vitro translation of mal1 RNA yielded a polypeptide of the predicted size of 15 kDa that was immunoprecipitable with an anti-rat liver fatty acid-binding protein antiserum. Based on the sequence analysis and antigenic properties of mal1, we conclude that it encodes a novel member of the lipid-binding protein family.

Amino Acid Sequence↗
PubMed1992

[Bilateral optic neuropathy following amiodarone therapy].

The amiodarone-induced optic neuropathy seldom occurs and so far, only 17 cases have been reported. These results are summarized here and the differences between them and the ischemic optic neuropathy are pointed out. We report on a 62-years-old patient who, when treated with amiodarone, developed an optic atrophy with slightly decreased visual acuity and severe visual field defects of the left eye. On the right eye we discovered shortly after discontinuing the amiodarone therapy, how, a mild swelling of the optic disc changed to an optic atrophy with analogous, irreversible changes of visual acuity and visual fields.

Amiodarone↗
PubMed1992

Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins.

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Amino Acid Sequence↗
PubMed1992

Overexpression of three ubiquitin genes in mouse epidermal tumors is associated with enhanced cellular proliferation and stress.

A mouse ubiquitin clone that recognizes multiple transcripts overexpressed in murine tumors compared to normal epidermis was isolated by differential screening of complementary DNA libraries from mouse squamous cell carcinomas. Coding region probes detected five ubiquitin transcripts. Oligonucleotides were designed for unique parts of three mouse ubiquitin gene complementary DNA clones. The overexpressed transcripts at 2.4, 2.8, 6.4, and 7.8 kilobases (kb) were detected by an oligonucleotide specific for a mouse UbC polyubiquitin clone. A 1.2-kb UbB overexpressed transcript was detected by an oligonucleotide for a mouse four-unit polyubiquitin, and a 0.7-kb UbA overexpressed transcript was recognized by an oligonucleotide for the mouse ubiquitin carboxyl-extension protein of 52 amino acids. All three classes of transcripts were induced in mouse skin by the hyperproliferative agent ethylphenyl propionate and by the tumor promoting agent 12-O-tetradecanoylphorbol-13- acetate. Heat shock of cultured keratinocytes induced both the 6.4- and 7.8-kb transcripts recognized by the UbC-specific oligonucleotide. Consistent with the overexpression of the ubiquitin transcripts, the level of free ubiquitin protein, as determined by Western analysis, was elevated in the tumors and proliferating epidermis as compared to normal epidermis. Our results indicate that the overexpression of ubiquitin genes could be related to a sustained state of proliferation and stress in the tumors compared to the normal resting epidermis.

Alkynes↗
PubMed1991

TGF-beta 1 and skin carcinogenesis: antiproliferative effect in vitro and TGF-beta 1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis.

The role of transforming growth factor-beta 1 (TGF-beta 1) in multisage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF-beta 1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D ("putatively initiated" cells) responded to TGF-beta 1 with slightly reduced sensitivity, and the papilloma-producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF-beta 1. Steady-state levels of TGF-beta 1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF-beta 1 mRNA in mouse epidermis. A constitutive overexpression of TGF-beta 1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Osteolytic activity of Walker carcinosarcoma 256 is due to parathyroid hormone-related protein (PTHrP).

The hypercalcemic Walker carcinosarcoma 256 of the rat is an animal model for humoral hypercalcemia of malignancy. Previous in vivo studies suggested the production of a parathyroid hormone-related protein (PTHrP) by the Walker tumor. Therefore, we have measured immunoreactive PTHrP in serum-free conditioned medium from cells derived from this tumor using an antibody raised against human PTHrP(1-34). Walker tumor cell conditioned medium (WCM) displaced 125I-hPTHrP(1-34) from the antibody in a dose dependent manner, whereas control medium contained no immunoreactive PTHrP. In contrast, we detected no secretion of immunoreactive rat parathyroid hormone (rat PTH) by the Walker tumor cells using a midregional radioimmunoassay for rat PTH. WCM stimulated adenylate cyclase in osteoblast like cells, the dose-response curve paralleling that of hPTHrP(1-34). This effect could be inhibited by the PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34) and by the addition of anti-hPTHrP(1-34) antibody. Bone resorbing activity of WCM in organ culture (calvaria of fetal rats) was not inhibited by indomethacin and glucocorticoids, suggesting a prostaglandin independent mechanism of osteoclast activation in this model.

Adenylyl Cyclases↗
PubMed1991

Identification of a cloned sequence activated during multi-stage carcinogenesis in mouse skin.

Differential screening of cDNA libraries made from chemically induced malignant mouse skin squamous cell carcinomas (SCC) identified three sequences, including one called mal2, that were upregulated in their expression at both the benign papilloma and malignant SCC stages. The mal2 plasmid cDNA clone (containing a 350 bp insert) was used to screen lambda phage cDNA libraries made from chemically induced SCCs. Two of the largest mal2-related cDNA inserts obtained from the phage libraries were sequenced. In addition a mal2-related genomic clone was obtained by hybridization probing of a mouse spleen genomic DNA library. The sequence of the genomic clone overlapped and was identical with both the mal2 plasmid and lambda cDNA clones. Identity was found between the mal2 cDNAs, the mal2 genomic sequence and the cDNA sequence for a mouse hyperproliferative keratin called K6. A synthetic oligonucleotide specific for the 3' untranslated region of the mal2 or keratin K6 gene was used in Northern analyses to demonstrate elevated steady-state levels of K6 keratin transcripts in SCCs induced by various protocols involving both chemical and ionizing radiation initiation of tumors as well as complete chemical and radiation carcinogenesis protocols. Metastatic lung lesions derived from SCCs generated by repeated doses of benzo[a]pyrene showed moderate levels of K6 keratin transcripts, whereas normal lung showed very low levels of K6 transcripts. The overexpression of the mal2 or keratin K6 gene in malignant SCCs was independent of the protocol, either chemical or radiation, that was used to induce the tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Differential gene expression during multistage carcinogenesis.

The use of the mouse skin multistage model of carcinogenesis has aided our understanding of critical target genes in chemical carcinogenesis. The mutagenic activation of the Harvey-ras proto-oncogene has been found to be an early event associated with the initiation of mouse skin tumors by the polycyclic aromatic hydrocarbon 7,12 dimethylbenz[alpha]anthracene and the pure initiator ethyl carbamate (urethane). In contrast to chemical initiation of mouse skin tumors, ionizing radiation-initiated malignant skin tumors have been shown to possess distinct non-ras transforming gene(s). Differential screening of cDNA libraries made from chemically initiated malignant skin tumors has been used to identify a number of cellular gene transcripts that are overexpressed during mouse skin tumor progression. These differentially expressed genes include beta-actin, ubiquitin, a hyperproliferative keratin (K6), a gene whose product is a member of a fatty acid or lipid-binding protein family, and a gene called transin or stromelysin. The overexpression of the stromelysin gene, which encodes a metalloproteinase that degrades proteins in the basement membrane, is hypothesized to play a functional role in malignant tumor cell invasion and metastasis. We believe that the cloning, identification, and characterization of gene sequences that are differentially expressed during tumor progression could lead to the discovery of gene products that either play functional roles in skin tumor progression or in the maintenance of various progressive tumor phenotypes.

3T3 Cells↗
PubMed1990

Expression of the N-myc proto-oncogene during the early development of Xenopus laevis.

The N-myc proto-oncogene is expressed in a wide range of tissues during mammalian embryogenesis. This observation, along with the oncogenic capacity of this gene, has led to the suggestion that N-myc plays an important role in early development. However, due to the complexity of the expression pattern and the difficulty of manipulating mammalian embryos, little progress has been made towards understanding the developmental function of this gene. To enable a more detailed analysis of the role of this gene in early development, a study of the Xenopus homologue of N-myc was undertaken. Xenopus N-myc cDNA clones were isolated from a neurula library using a murine N-myc probe. Analysis of the timing of expression of N-myc mRNA and of the distribution of N-myc protein during Xenopus development indicate that this gene may be playing an important role in the formation of a number of embryonic structures, including the nervous system. N-myc is initially expressed as a maternal RNA, but this mRNA is degraded by the gastrula stage of development. Zygotic expression does not commence until late neurula. Examination of the distribution of the N-myc protein by whole-mount immunohistochemistry indicates that the early embryonic expression occurs in the central nervous system, the neural crest, the somites and the epidermis. Later expression is mostly within the head and somites. Specific structures within the head that express the protein include the eye, otic vesicle, fore and hindbrain and a number of cranial nerves. The results demonstrate that while N-myc is expressed in the developing nervous system of Xenopus, the timing of expression indicates that it is unlikely to be involved in regulation of the very first stages of neurogenesis.

Animals↗