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M Engel

Publications and source records attributed to M Engel.

At least 55 records · Page 3Linked to original sources
PubMed1996

Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit.

IL-12 enhances IFN-gamma production by NK cells and T lymphocytes and plays a pivotal role in the development of Th1 cells. Bioactive IL-12 (p70) is composed of two subunits, p35, which is only secreted as part of the p70 dimer, and p40, which can also be secreted by itself. The IL-12 subunits are encoded by two separate genes. Therefore, we studied to what extent the expression of each of the subunits contributes to the regulation of IL-12 production. We found that in LPS-stimulated whole blood and purified monocytes, p70 and p40 production are enhanced by IFN-gamma and inhibited by IL-10 and IL-4. However, IFN-gamma and IL-10 had stronger effects on p70 production than on p40 production, and IL-4 affected p40 production more strongly. Concomitantly, in all experimental conditions tested, p40 production greatly exceeded p70 production, suggesting that p35 expression was limiting. Analysis of p35 and p40 mRNA expression by PCR confirmed this notion. Resting purified monocytes expressed neither p40 nor p35 mRNA. The effects of IFN-gamma, IL-10, and IL-4 on the p35 mRNA expression in LPS-stimulated purified monocytes were similar to the effects of these cytokines on p70 protein production, and the p40 mRNA expression corresponded to p40 protein production. Our results imply that production of the two IL-12 subunits is differently regulated for each subunit, mainly at the level of mRNA expression, and that the level of bioactive IL-12 production in monocytes in response to LPS and cytokines is determined by the level of p35 expression.

Antibodies, Monoclonal↗
PubMed1996

Risk factors and dose-effect relationship for osteoradionecrosis after hyperfractionated and conventionally fractionated radiotherapy for oral cancer.

A high frequency of osteoradionecrosis after hyperfractionated radiotherapy (RT) of head and neck tumours led to a detailed analysis of risk factors in the dental, surgical, and radiotherapeutic areas. 168 patients with oral cancer were analysed retrospectively. 19% of them had been irradiated primarily and 81% postoperatively. 116 patients received a total dose mostly ranging from 60 Gy to 70 Gy to the ICRU 29 reference point (daily single dose 2 Gy). 52 patients were treated hyperfractionally with two daily fractions of 1.2 Gy per day, 4 h minimum apart and a total dose 82.8 Gy. Dental findings could be evaluated in 126 patients. Factors were checked for prognostic significance for osteoradionecrosis (ORN). Dose dependency was computed using a PROBIT analysis. Dental status before radiotherapy was generally poor (mean 11/32 teeth present, of these 1 was dead, 2.4 carious, 2.4 loose, 0.3 destroyed). On average, six teeth (range 0-27 teeth) had to be extracted. In one-third of the patients bone surgery was necessary. ORN occurred in 8.6% of the patients treated conventionally but in 22.9% of those treated hyperfractionally (p = 0.029). Biologically effective dose (p = 0.032) and deep paradontitis (p = 0.034) proved to be significant risk factors for ORN. PROBIT analysis showed a steadily rising dose dependency of the ORN frequency after conventional radiotherapy. Using total doses up to 70 Gy the frequency of ORN was 8.6%. Dose escalation using hyperfractionation led to an intolerable ORN frequency (22.9%) where a short interfraction interval was a significant factor. The use of this dose fractionation was therefore discontinued in 1992.

Dose-Response Relationship, Radiation↗
PubMed1995

A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Amino Acids↗
PubMed1995

Characterization of the human nm23-H2 promoter region and localization of the microsatellite D17S396.

The transcription of human nm23 genes (nm23-H1, nm23-H2) is involved in suppression of tumor metastasis or tumor progression. Therefore the characterization of transcriptional regulatory mechanisms for both nm23 genes is very important. In this study we have isolated and analyzed the 5'-flanking region of the human nm23-H2 gene and estimated the distance to 4 kb between nm23-H2 and nm23-H1 genes. We localized the known microsatellite D17S396 within this region. Furthermore the identification of possible binding sites for MYC proteins and additionally the NM23-H2 protein itself (the transcription factor PuF for c-myc gene activation) is of importance with respect to possible para- and autoregulatory interactions. A comparison of the promoter sequences of both human nm23 genes revealed no significant sequence homology.

Animals↗
PubMed1995

Stress causes induction of MAP kinase-specific phosphatase and rapid repression of MAP kinase activity in Drosophila.

Heat shock and chemical stress induce activation of heat shock (stress) genes and synthesis of heat shock proteins. It is not yet fully understood which molecular mechanism leads to activation. Probably denatured proteins play an important role in activating the transcription factor (HSF), but on the other hand there are many hints that a phosphorylation event is involved, too. During a search for a possible signal transduction system in Drosophila Schneider 2 cells we analysed the response of the mitogen-activated protein (MAP) kinase after stress and its regulation by phosphatases. We show here that stress activates a MAP kinase-specific phosphatase in Drosophila and inhibits MAP kinase activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

[Proton spin tomography of the orbit in post-traumatic motility disorders].

AIM: To analyse the value of MRI for the assessment of posttraumatic disturbances of eye motility. MATERIAL AND METHODS: We analysed retrospectively the results of 38 MR examinations of the orbit in 31 patients with posttraumatic motility impairment with preserved visus. 18 patients underwent MRI preoperatively. From this group 5 patients were additionally examined postoperatively. Another 5 patients who had not been examined prior to surgery were controlled postoperatively. Hence, a total of 10 patients with persisting disturbances was examined postoperatively. 8 patients who underwent MRI were not treated by surgery because of only minor disturbances. In all patients conventional radiography of the orbit was performed prior to MRI. Additional CT imaging was carried out in 12 patients. RESULTS: In 18 patients examined preoperatively we found displacement of orbital fat tissue, displacement and entrapment of orbital muscles, swelling of muscles and oedema in retrobulbar fat tissue. 10 patients from the postoperative group exhibited remaining prolapsed fat tissue, oedema in fat tissue and/or swelling of muscles. 8 patients had only small soft tissue changes which did not require surgery. CONCLUSION: Since it can image soft tissue precisely, MRI provides the decisive information in the assessment of motility impairment of the eye. In case of isolated orbital fracture with motility impairment, CT is not absolutely necessary for surgical therapy.

Adolescent↗
PubMed1994

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

Animals↗
PubMed1994

Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro.

We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK's from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

Amino Acid Sequence↗
PubMed1994

Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung.

We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

Squamous cell carcinoma of the lung: does the nm23 gene expression correlate to the tumor stage?

We examined the nm23 gene activity in 30 human squamous cell carcinomas of the lung using the Northern blot analysis. Matched healthy lung tissue was available from 10 patients. 26 tumor samples were obtained after resection treatment between 1986 and 1990, 4 additional samples of advanced stages from autopsy. We found a significant increase of nm23 expression towards advanced stages of squamous cell carcinoma (IIIa vs. I: p < 0.015, IV vs. II: p < 0.043, IV vs. I: p < 0.043). Furthermore in stage I and II, poorly differentiated squamous cell carcinomas contained significantly more nm23 mRNA than did moderately differentiated ones (p < 0.027). We could also demonstrate an inverse correlation between the levels of nm23 mRNA and disease-free survival after resection treatment (p < 0.016, chi 2-test for trend). Thus the nm23-gene activity correlates to the tumor stage and the grade of differentiation of the squamous cell carcinoma of the lung, and is therefore of prognostic relevance. It might be possible to describe the biological behaviour of the individual tumor more accurately by the additional measurement of the nm23 mRNA.

Carcinoma, Squamous Cell↗
PubMed1994

Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue.

Using immunocytochemistry and in situ hybridization histochemistry, we have investigated in embryonic and postnatal rat nervous tissue the localization and cellular sites of synthesis of glypican, a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. Glypican immunoreactivity is present in the marginal layer (prospective white matter) and in the dorsal root entry zone of E13-16 spinal cord, as well as in the optic nerve and retina at this stage, but does not appear at significant levels in brain until approximately E19. The proteoglycan shows a wide distribution in grey matter and axonal projections of postnatal brain, including the hippocampal formation, the parallel fibers of cerebellar granule cells, and in the medulla and brainstem. Northern analysis demonstrated high levels of glypican mRNA in brain and skeletal muscle, and in rat PC12 pheochromocytoma cells. In situ hybridization histochemistry showed that glypican mRNA was especially prominent in cerebellar granule cells, large motor neurons in the brainstem, and CA3 pyramidal cells of the hippocampus. Our immunocytochemical and in situ hybridization results indicate that glypican is predominantly a neuronal membrane proteoglycan in the late embryonic and postnatal rat central nervous system.

Amino Acid Sequence↗
PubMed1994

Kinetics of inhibition by tyrphostins of the tyrosine kinase activity of the epidermal growth factor receptor and analysis by a new computer program.

The kinetics of inhibition of the epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase (TK) activity by erbstatin, tyrphostins, and lavendustin derivatives were studied in a system that employs poly(Glu6Ala3Tyr) (GAT) and ATP as substrates, after preactivation with EGF. All data were analyzed for computer best-fit curves by a program that was written for this purpose and is available upon request to those interested. The inhibition kinetics followed a sequential, Bi-Bi, rapid equilibrium, random mechanism, the mechanism of the EGFR-TK. Erbstatin and a few tyrphostins that contain a 3,4-dihydroxy-(cis)-cinnamonitrile [1-(3',4'-dihydroxyphenyl)-2-nitriloethene] group were found to be pure competitive inhibitors with respect to both substrates of the kinase reaction, i.e., GAT and ATP. Two tyrphostins, each containing an additional dihydroxyphenyl group in the alpha-position, were found to be pure competitive inhibitors with respect to GAT and noncompetitive (or mixed-competitive) inhibitors with respect to ATP. A lavendustin derivative with a 2,5-dihydroxyphenyl ring and a lavendustin derivative with a 3,4-dihydroyphenyl ring were also found to be competitive inhibitors with respect to both ATP and GAT. Various possible modes of binding at the EGFR-TK active center for the tyrphostins studied are proposed and the significance of the present findings, as well as the interpretations of computer analyses of kinetic data, is discussed.

Catechols↗
PubMed1993

High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages.

Expression of the candidate metastasis-suppressor gene nm23-H1 has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To determine whether the nm23 genes could have a metastasis-suppressor function in non-small-cell lung carcinoma (NSCLC), pulmonary sarcoma and carcinoids, we analysed both nm23-HI and nm23-H2 mRNA levels in 37 tumor samples obtained from patients who underwent potentially curative resection between 1986 and 1990, and in 4 metastatic tumors obtained from autopsy. As compared to corresponding healthy lung parenchyma, both nm23-HI and nm23-H2 transcript levels were elevated in 37 of 41 tumors. The increases in nm23 mRNA expression were stronger in advanced stages of squamous-cell carcinoma, large-cell carcinoma, sarcoma and carcinoids than in early stages of the respective tumor types. Within stages I and II of squamous-cell carcinoma, significantly higher nm23 mRNA levels were found in poorly differentiated tumors than in moderately differentiated ones. Moreover, an inverse correlation between nm23 expression and disease-free survival of the patients was observed. In conclusion, our results indicate that the increased nm23 expression in the analysed tumors is not consistent with the proposed metastasis-suppressor function, but the 2 nm23 genes nevertheless may be implicated in the mechanism of tumor progression.

Adenocarcinoma↗
PubMed1993

A rapid method to determine the orientation of blunt end ligated polymerase chain reaction products.

Subcloning of polymerase chain reaction (PCR) fragments is often performed via blunt end ligation after previous Klenow fragment exonuclease treatment. Since insert-specific primers are at hand from the original amplification step, we used a simple PCR-based assay to determine the insert orientation of the recombinants. After purification of enriched plasmids, small aliquots were tested performing standard PCR reactions using a plasmid primer directed towards the cloning site and one of the insert specific primers, respectively. Only the distant insert primer yields a PCR product which can be visualized after gel electrophoresis. In this way both the insert orientation in the vector and the correct size of the cloned fragment is determined rapidly without sequencing or restriction fragment analysis.

Animals↗
PubMed1993

[The dynamics of blood composition changes in leukocyte filtration during cardiopulmonary bypass. Preliminary results].

Activation of leukocytes (especially neutrophils) during cardiopulmonary bypass (CPB) causes a reperfusion injury through the release of oxygen free radicals and formation of microvascular occlusions. Leukocyte filtration during CPB could solve these drawbacks. We investigated the selectivity of leukocyte filtration by fifteen patients undergoing coronary artery bypass surgery. Leukocyte depleted patients (n = 8) had a leukocyte filter incorporated in the bypass circuit. We evaluated the number of lymphocytes, neutrophils and platelets as well as hematocrit values at following time points: a) before CPB, b) 15 min after the start of CPB, c) before the administration of protamin and d) 3 hours after the CPB. No statistically significant changes of hematocrit values, lymphocyte or platelet counts between control and leukocyte depleted group of patients were observed. However, the use of leukocyte filter caused significant decrease in number of circulating neutrophils at 15 min after begin of CPB (1.20 +/- 0.17 x 10(9)/l for leukocyte depleted patients and 1.90 +/- 0.42 x 10(9)/l for control group of patients, p < 0.05). Our temporary results indicate the selectivity of leukocyte filtration during the cardiopulmonary bypass.

Adult↗
PubMed1993

[4-fragment fracture of the proximal upper arm].

Four-fragment fractures have to be accorded great importance because they are so severe. The morphology allows differentiation of two types, the dorsolateral fracture type and the impacted fracture type. Over a 4-year time span, 13 of these fractures were treated with minimal osteosynthesis. This involve the open anatomical reduction and allogeneic spongiosa grafting and retention of the fracture with screws and, if necessary, K-wires and tension banding. After an average of 26.3 months the shoulder function was evaluated using the CONSTANT score. The dorsolateral fractures were scored of 87.2 while the impacted fractures were scored at 88.8. No necrosis of the humeral head was found. These good results show that after four-fragment fractures primary reconstruction of the humeral head should be preferred over primary prosthetic replacement.

Adult↗