Biomedical subjects

E Uhlmann

Publications and source records attributed to E Uhlmann.

42 records · Page 3Linked to original sources

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↗

PubMedDate not supplied

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells.

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.

3' Untranslated Regions↗

PubMedDate not supplied

Peptide nucleic acids (PNA) and PNA-DNA chimeras: from high binding affinity towards biological function.

Oligonucleotide analogs are of major interest as tools in molecular biology, as diagnostics, and as potential pharmaceuticals which bind in a predictable way to certain nucleic acid target sequences, aiming at the inhibition of expression of disease-causing genes. One of the most promising nucleic acid mimetics are the peptide - or polyamide- nucleic acids (PNA) which bind with higher affinity to DNA and RNA than natural oligonucleotides. In these non-ionic PNAs, the entire sugar-phosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure. A unique property of PNA is its ability to displace one strand of a DNA double-helix. This strand displacement process, which is inefficient with DNA, is supported by the formation of an unusually stable internal (PNA)2 x DNA triple helix. The combination of PNA and DNA in one molecule results in PNA/DNA chimeras with new properties. They show improved aqueous solubility compared to pure PNAs due to their partially negatively charged structure. Furthermore, the cellular uptake of the chimeras is better than of pure PNAs. In contrast to PNA, the chimeras bind exclusively in the antiparallel orientation under physiological conditions. The binding affinity is generally stronger when the PNA/DNA chimeras are hybridized to RNAthan to DNA, whereby the strength of binding strongly depends on the PNA: DNA ratio. Most interestingly, PNA/DNA chimeras are recognized as substrates by various nucleic acid processing enzymes, and consequently can also assume biological functions, such as a primer function for DNA polymerases. Pure PNA cannot induce RNase H cleavage of target RNA, which is believed to support the biological efficacy of antisense agents. However, DNA-PNA chimeras are able to stimulate cleavage of the target RNA by RNase H upon formation of an RNA x chimera duplex.

Base Sequence↗