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1.
Johan B. Ubbink W. J. Hayward Vermaak Siegbert Bissbort 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,565(1-2)
A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiol-specific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory. 相似文献
2.
Siegbert Biekmann J. Feierabend 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(3):268-279
According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis. 相似文献
3.
Siegbert Hummel 《Ethnos》2013,78(1-2):31-44
This article examines the special biases, social ambiguities and methodological problems characterizing ethnographic research among fieldworkers with cultural orientations vaguely or partially congruent with their hosts. Information for this discussion derives from a recent field study of agrarian adaptations in northeastern Finland conducted by an anthropologist of third‐generation immigrant Finnish‐American background. This context is used to address broad issues regarding detachment and objectivity, limits upon shared experience and meaning, and interpretive dilemmas facing the ethnographic enterprise generally. 相似文献
4.
Siegbert Rieg Benjamin Meier Eva Fähnrich Anja Huth Dirk Wagner Winfried V Kern Hubert Kalbacher 《BMC microbiology》2010,10(1):61
Background
Members of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human α-defensins human neutrophil peptides (HNPs) 1-3, human β-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine β-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated. 相似文献5.
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Two lactate dehydrogenase (LDH) mutations were recovered independently among offspring of ethylnitrosourea-treated male mice by screening for alterations of isoelectric focusing pattern in liver homogenates. Investigations of physicochemical and kinetic properties of the mutant enzymes indicated that the mutant traits resulted from point mutations at theLdh-1 structural locus. Therefore, the new alleles were designatedLdh-1
a-m5Neu andLdh-1
a-m6Neu, respectively. Both mutant alleles code for proteins which exhibit an altered stability to heat, in addition to changes in isolectric focusing pattern and a reduction in anodal electrophoretic mobility. While LDH-Aa-m5Neu proteins are markedly less heat stable, LDH-Aa-m6Neu proteins are more heat stable than the wild-type enzyme. Furthermore, a small elevation ofK
m for pyruvate, a slightly reduced inhibition by high pyruvate concentrations, and a slight acidic shift of the pH activity profile distinguish LDH-Aa-m6Neu from both wild-type and LDH-Aa-m5Neu enzymes. Significant alterations of LDH activity were detected in some tissues from LDH-Aa-m5Neu individuals but not in those from LDH-Aa-m6Neu animals. Erythrocytes and blood of LDH-Aa-m5Neu mutants revealed activity levels which were reduced by approximately 6 and 13% compared with those of wild types in heterozygous and homozygous individuals, respectively. In addition, an elevation of approximately 6% in LDH activity was found in skeletal muscle in homozygous mutants. Consistent with the unaltered or only slightly altered LDH activity in tissues, the genetic as well as the physiological characterization yielded no easily detectable effects from either mutation on metabolism or fitness of the affected individuals.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities. 相似文献
8.
Walter Pretsch Bimal Chatterjee Jack Favor Siegbert Merkle Rodica Sandulache 《Mammalian genome》1998,9(2):144-149
Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in
whole blood have been recovered. The mutant line Ldh1
a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1
a7Neu, Ldh1
a11Neu, and Ldh1
a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the
number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical
and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1
a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix
αD, which is involved in the coenzyme-binding domain. Ldh1
a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which
may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1
a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations,
which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of
the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1
a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation
Ldh1
a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations
has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical
and physiological alterations.
Received: 3 July 1997 / Accepted: 30 September 1997 相似文献
9.
Roessler M Rollinger W Mantovani-Endl L Hagmann ML Palme S Berndt P Engel AM Pfeffer M Karl J Bodenmüller H Rüschoff J Henkel T Rohr G Rossol S Rösch W Langen H Zolg W Tacke M 《Molecular & cellular proteomics : MCP》2006,5(11):2092-2101
The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate. 相似文献
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