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E Uhlmann

Publications and source records attributed to E Uhlmann.

18 recordsLinked to original sources
PubMed1995

Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Adenovirus E1A Proteins↗
PubMed1995

Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock.

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, we blocked the expression of c-Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c-Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker.

Animals↗
PubMed1995

Inhibition of c-Fos expression in the UV-irradiated epidermis by topical application of antisense oligodeoxynucleotides suppresses activation of proliferating cell nuclear antigen.

Induction of c-fos protooncogene expression following exposure of mammalian skin to UV irradiation suggests an involvement in UV-induced alterations of epidermal cell proliferation and viability. In the present study we have investigated whether topically administered c-fos antisense oligodeoxynucleotides (ODNs) inhibit c-fos activation in the UV-exposed rat skin and thereby modulate the delayed increase in cellular proliferative activity. The accumulation of c-Fos immunolabeled nuclei in the epidermis was almost completely blocked 18 h post-irradiation by topical treatment with the c-fos antisense ODN. The co-expression of c-Jun was not affected and a random sequence control ODN was ineffective. Epicutaneous application of fluorescein-labeled ODNs revealed penetration into the underlying epidermis. The appearance of nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA) 18 h after UV exposure was significantly suppressed in the epidermis treated with c-fos antisense ODNs. In vitro PCNA is involved in both DNA repair synthesis and DNA replication, and the expression of PCNA mRNA is increased after UV irradiation. Thus, it may be speculated that UV-induced c-Fos transcription factor may be linked to repair of photodamaged DNA and/or cell cycle progression by trans-activating PCNA gene expression.

Administration, Topical↗
PubMed1995

Expression of c-Fos and c-Jun in the cornea, lens, and retina after ultraviolet irradiation of the rat eye and effects of topical antisense oligodeoxynucleotides.

AIMS: Immunohistochemical techniques were used to investigate c-Fos and c-Jun proto-oncogene expression in the cornea, lens, and retina after ultraviolet irradiation of the rat eye. METHODS: Eyes of anaesthetised rats were exposed to 1.5 J/cm2 of ultraviolet radiation (280-380 nm). Animals were perfused 1, 6, or 24 hours after irradiation and tissue sections were incubated with specific antiserum to c-Fos and c-Jun, respectively. RESULTS: Non-irradiated contralateral eyes displayed no c-Fos and c-Jun immunoreactivity. One and 6 hours after ultraviolet exposure numerous c-Fos and c-Jun immunopositive nuclei were observed mainly in the epithelial cell layers of the cornea and the lens epithelium. Scattered labelled nuclei were detectable in the retinal ganglion cell layer and the inner nuclear layer. Twenty four hours after irradiation c-Fos and c-Jun protein expression returned to near control levels. Histological signs of ultraviolet damage (for example, chromatin condensation, nuclear fragmentation) were first recognisable in the corneal epithelium 6 hours after irradiation and became more apparent at later times. CONCLUSION: Thus, the rapid and sustained activation of c-Fos and c-Jun expression in the eye after single ultraviolet exposure may represent the molecular mechanism underlying ultraviolet induced photodamage and initiation of cell death. Furthermore, topical application of a c-fos antisense oligodeoxynucleotide to the ultraviolet exposed rat eye inhibited the increase in c-Fos expression in the cornea, suggesting therapeutic activity of antisense drugs in corneal malignant and infectious diseases.

Administration, Topical↗
PubMed1995

Inhibition of viral growth by antisense oligonucleotides directed against the IE110 and the UL30 mRNA of herpes simplex virus type-1.

In the present work we elucidate that the identification of active sequences for a given target is one of the principle hurdles of antisense oligonucleotide therapeutics. A number of 100 oligonucleotides directed against different target genes of HSV-1 and different locations within those genes were screened for antiviral activity. To facilitate comparison, the same length and the same chemical modification were used for all oligonucleotides: 20mers with two phosphorothioate linkages at both the 5'- and the 3'-end. No sequence-independent effects were observed with this type of modification. Surprisingly, only six oligonucleotides did show significant antiviral activity, the most active one (#6) being directed against the translation initiation site of IE 110.

Base Sequence↗
PubMed1994

Inhibition of c-Fos protein expression in rat spinal cord by antisense oligodeoxynucleotide superfusion.

Peripheral noxious stimulation leads to a rapid and transient expression of c-fos, c-jun and other immediate-early genes (IEGs) in the spinal cord. However, the role of IEG encoded transcription factors in plasticity of spinal neurons remains speculative. In the present study we have shown that superfusion of rat spinal cord with antisense oligodeoxynucleotides complementary to c-fos mRNA suppresses heat-induced c-Fos protein expression without affecting other members of the Fos and Jun family, thus providing a technique to determine the function of IEGs in vivo.

Animals↗
PubMed1994

Differential regulation of c-fos, fosB, c-jun, junB, bcl-2 and bax expression in rat skin following single or chronic ultraviolet irradiation and in vivo modulation by antisense oligodeoxynucleotide superfusion.

Single ultraviolet (u.v.) irradiation of mammalian cells in culture evokes the transcriptional activation of various proto-oncogenes, among them members of the fos/jun family which are known to play an important role in cell proliferation and differentiation. u.v. exposure of mammalian skin results in growth arrest and cell death followed by hyperproliferation of epidermal cells. To obtain information in vivo about a possible relationship between u.v.-induced proto-oncogene expression and cellular alterations, we have analysed the expression of c-fos, fosB, c-jun, junB, bcl-2 and bax in rat epidermis after single and chronic u.v. irradiation. We present data demonstrating that the transcripts of these genes are constitutively expressed in the epidermis and that expression is differentially modulated by u.v. exposure. Single u.v. irradiation causes a rapid and sustained increase in c-jun, junB and c-fos mRNA and a decline in bcl-2 transcripts, whereas expression of bax remained unchanged. c-Fos and c-Jun immunoreactivity was localized throughout the epidermal cell layers 1.5 h after single irradiation, but restricted to basal cells at 48 h suggesting an involvement in both u.v.-induced apoptosis and hyperproliferation. 48 h after chronic exposure a significantly higher induction and a totally different pattern of epidermal proto-oncogene expression was detectable which may be associated with malignancy. Superfusion of rat skin with c-fos antisense oligodeoxynucleotides inhibited the increase in c-Fos immunolabeled epidermal cells 1.5 h after single u.v. irradiation demonstrating that antisense oligodeoxynucleotides are capable of penetrating mammalian skin and modulating the u.v. response in vivo. However, suppression of the early c-Fos activation did not significantly affect the formation of sunburn cells in the u.v.-exposed epidermis. Thus, c-Fos does not seem to play a major role in u.v.-induced apoptosis or other members of the fos/jun family may compensate for a loss in c-Fos.

Animals↗
PubMed1990

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.

Aminobutyrates↗
PubMed1988

An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA.

A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented. The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure. The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes. Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length. Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments. By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size. The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.

Base Sequence↗
PubMed1988

Gene synthesis.

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Cloning, Molecular↗
PubMed1988

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein.

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.

Amino Acid Sequence↗
PubMed1988

The effect of chronic bradycardial pacing on the oxidative capacity in rabbit hearts.

Blood flow and oxygen consumption were estimated in isolated hearts from control rabbits or animals with chronic bradycardia achieved by transvenous atrial pacing for 4 weeks; these parameters were related to ultrastructural changes. Chronic reduction of heart frequency to about 50% of control values resulted in an increased capillary/fibre ratio (1.43 +/- 0.07 in paced, 1.26 +/- 0.07 in control hearts), increased capillary density (2339 +/- 148 vs 1897 +/- 95) and increased volume density of mitochondria (36.36 +/- 1.28% vs 31.38 +/- 1.7%), in paced and control hearts, respectively, with no signs of heart hypertrophy (fibre diameters 28.5 micron in both groups). Maximal blood flow (3 ml.g-1.min-1) was similar in paced and control hearts. Maximal oxygen consumption (achieved by a gradual increase in preload and increase in force of contraction by noradrenalin infusion) in paced hearts was significantly higher than in control hearts and was achieved by a higher oxygen extraction. This could be due both to the increased volume density of mitochondria and to a more homogeneous distribution of flow through an enlarged capillary bed. The increased oxidative capacity induced by chronic bradycardial pacing can explain improved maximal cardiac work found under these conditions previously in vivo.

Animals↗
PubMed1987

Relationship between the size of the capillary bed and oxidative capacity in various cat skeletal muscles.

The size of the capillary bed, assessed by capillary density (CD), capillary per muscle fibre ratio (C/F), total capillary length, surface area and volume was related to the oxidative capacity, assessed by the volume density of mitochondria and VO2max in cat muscles with a different composition of glycolytic and oxidative fibres: predominantly glycolytic gracilis, purely oxidative soleus and gracilis transformed towards oxidative by chronic low frequency (10 Hz) electrical stimulation. Maximal blood flow and lactate output were measured in the muscles during isometric contractions. When capillary supply was estimated by C/F ratio, there was a close correlation between various parameters only in stimulated gracilis. The combined data of all muscles showed a significant correlation between the total volume of mitochondria, VO2max and total capillary surface area. Capillary volume showed a tight correlation with maximal blood flow in both control and stimulated gracilis, but not in soleus. Maximal blood flow was correlated with VO2max in oxidative muscles (stimulated gracilis and soleus) but not in control glycolytic gracilis. Moreover normal gracilis did not show any relationship between the volume density of mitochondria and the size of the capillary bed. The latter was inversely correlated with the output of lactate which was greater in muscles with a lower C/P ratio. The data on gracilis indicates that the capillary bed can adapt to the increased demand for oxygen and a greater oxidative capacity induced by long-term activity imposed on a glycolytic muscle, while it may be more important for the removal of lactate in the glycolytic muscles under their normal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45.

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.

Amino Acid Sequence↗
PubMed1987

Relationship between mitochondria and oxygen consumption in isolated cat muscles.

1. Oxygen consumption, mitochondrial content and composition, intracellular lipid stores and fibre size were studied in isolated cat muscles: predominantly glycolytic gracilis, purely oxidative soleus and gracilis transformed into an oxidative muscle by chronic low-frequency (10 Hz) electrical stimulation. 2. Oxygen consumption in control gracilis at rest (0.303 +/- 0.050 ml O2 min-1 100 g-1; mean +/- S.E. of mean) was three to five times lower than in either stimulated gracilis (1.16 +/- 0.40) or soleus (1.57 +/- 0.56); it was about two times lower during maximal contractions in control gracilis (5.15 +/- 0.24) than in either stimulated gracilis (11.6 +/- 2.0) or soleus (9.34 +/- 0.78). 3. The volume density of mitochondria in control gracilis (2.75 +/- 0.12%) was half that of soleus (6.23 +/- 0.76) and only one-third that of stimulated gracilis (8.35 +/- 0.71). Subsarcolemmal mitochondria represented a significantly smaller fraction of the total mitochondrial volume in control gracilis than in either soleus or stimulated gracilis. 4. The surface area of inner and outer mitochondrial membranes per unit volume of mitochondria ranged from 23.4 to 26.1 and from 14.0 to 16.5 m2 cm-3, respectively. Mean values of these variables were not significantly different among experimental groups. 5. The volume density of the intracellular lipid stores in control gracilis (0.232 +/- 0.041%) was one-fourth of that in stimulated gracilis (0.860 +/- 0.12) and one-fifth of that in soleus (1.17 +/- 0.27). 6. The fibre cross-sectional area was 1670 +/- 260 micron 2 in control gracilis, 2250 +/- 280 in stimulated gracilis and 2390 +/- 110 in soleus. The difference was statistically significant only between control gracilis and soleus. 7. There was a significant correlation between the volume density of mitochondria and maximal oxygen consumption for all three muscles combined. 8. It was found that mitochondrial structure was similar in muscles with different oxidative capacities and that equal amounts of mitochondria consumed equal amounts of oxygen under limiting conditions of maximal in vivo respiration.

Animals↗
PubMed1987

Chemoenzymatic synthesis of genes encoding medium-sized polypeptides by use of only one synthetic oligonucleotide.

A novel strategy for the synthesis of genes encoding medium sized polypeptides from only one synthetic oligodeoxynucleotide is outlined. A 140-mer oligodeoxynucleotide forming a hairpin structure at its 3'-end has been synthesized and successfully used in the construction and cloning of a gene coding for salmon calcitonin-gly(33). Employing this "one oligonucleotide - one gene" approach the manual work required for oligodeoxynucleotide synthesis is reduced to a minimum.

Amino Acid Sequence↗
PubMed1980

Recent progress in oligonucleotide synthesis.

It was found that some improvements in oligoribo- and oligo-2'-deoxyribonucleotide synthesis via the phosphotriester approach could be achieved using the p-nitrophenylethyl group for phosphate protection due to its stability in the condensation step and its clean removal of DBU and DBN respectively. Recent results will prove the synthetic advantages over known procedures.

Bridged Bicyclo Compounds↗