PubMed Health⌕ Search

Biomedical subjects

R Geiger

Publications and source records attributed to R Geiger.

At least 73 records · Page 4Linked to original sources
PubMed1988

Determination of leukocyte elastase-inhibitor complexes and leukocyte neutral proteinase inhibitor by enzyme immunoassays. Leukocyte elastase-inhibitor complexes in porcine blood, III.

Sensitive enzyme immunoassays for the determination of total leukocyte neutral proteinase inhibitor and polymorphonuclear elastase-leukocyte neutral proteinase inhibitor complexes are described. The usable ranges of the standard curves were from 80 ng/l to 10 micrograms/l. The relative intra-assay coefficients of variation of the tests were between 2 and 4%, and the inter-assay coefficients of variation between 4 and 10%. In vitro and in vivo studies were performed with septic pigs and isolated leukocytes. The results show that leukocyte neutral proteinase inhibitor can be used in pigs as a parameter for the development of a septicaemia.

Animals↗
PubMed1988

Human urinary kallikrein--amino acid sequence and carbohydrate attachment sites.

The amino acid sequence of human urinary kallikrein was determined by protein-chemical methods. The peptide chain contains 238 amino acid residues. Ten half-cystine residues form five disulfide bridges. Six carbohydrate side chains could be identified, three of them are N-glycosidically linked to asparagine which is present in the acceptor sequon Asn-Xaa-Thr (or -Ser) and three are O-glycosidically linked to two serine residues and one threonine residue, respectively. Comparison of the primary structure of human urinary kallikrein with that of other serine proteinases especially porcine kallikrein and rat kallikrein shows a high degree of homology. IVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTA AHCISDNYQLWLGRHNLFDDENTAQFVHVSESFPHPGFN MSLLENHTRQADEDYSHDLMLLRLTEPADTITDAVKVVE LPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKIL PNDECEKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLM CDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS.

Amino Acid Sequence↗
PubMed1988

The application of a photon-counting camera in very sensitive, bioluminescence-enhanced detection systems for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, II.

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots was described recently. This method utilizes antibodies conjugated with alkaline phosphatase. Alkaline phosphatase releases D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light produced is measured with a very sensitive photon counting camera (Argus 100), permitting visualization and localization of the alkaline phosphatase-conjugated antibodies on nitrocellulose sheets. Under non-optimized conditions the limit of detection is at present 5 to 500 fg of protein (rabbit immunoglobulin G), corresponding to 30 to 3 amol. The method is therefore 10(5) times more sensitive than other used at present.

DNA↗
PubMed1987

Biochemistry of human tissue (urinary) kallikrein.

Human urinary kallikrein was purified by gel filtration on Sephacryl S-200 and affinity chromatography on aprotinin-Sepharose, followed by ion exchange chromatography on DEAE-Sepharose. Thus an enzyme preparation with a specific activity of 1100 U/mg protein (substrate: AcPheArgOEt) was obtained. In dodecyl sulfate electrophoresis two protein bands with apparent molecular weights of 41 kDa (form B) and 34 kDa (form A) have been separated. On isoelectric focusing different protein bands with isoelectric points between 3.75 and 4.25 were found. Both forms (form A and B) could be separated by gel filtration on Sephadex G-100 and characterized by dodecyl-sulfate electrophoresis and isoelectric focusing. The kinetic constants for the kallikrein-catalyzed hydrolysis of AcPheArgOEt and DValLeuArgNan were determined. The Ki value for the tissue kallikrein aprotinin complex was measured as well as the bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 9 +/- 2 1 x mol-1 x min-1). The amino acid composition and the amino acid sequence of active human tissue (urinary) kallikrein is presented.

Amino Acid Sequence↗
PubMed1987

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes.

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.

Animals↗
PubMed1987

Synthesis and characterization of luciferin derivatives for use in bioluminescence enhanced enzyme immunoassays. New ultrasensitive detection systems for enzyme immunoassays, I.

Derivatives of luciferin, D-luciferin methyl ester, D-luciferyl-L-phenylalanine, D-luciferyl-L-N alpha-arginine, D-luciferin-O-sulphate and D-luciferin-O-phosphate, were synthesized for use as highly sensitive substrates for enzyme assays. The luciferin derivatives were characterized by ultraviolet and fluorescence spectrophotometry, by amino acid analysis and by fast atom bombardement mass spectrometry. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants were determined for the following enzyme/substrate pairs: D-luciferin methyl ester/carboxylic esterase, D-luciferyl-L-phenylalanine/carboxypeptidase A, D-luciferyl-L-N alpha-arginine/carboxypeptidase B, D-luciferin-O-sulphate/arylsulphatase, D-luciferin-O-phosphate/alkaline phosphatase. All compounds proved to be acceptable substrates for the respective enzymes, D-luciferin-O-phosphate being accompanied by an especially high turnover number (kcat = 1010 s-1) with alkaline phosphatase.

Chromatography, High Pressure Liquid↗
PubMed1987

Bioluminescence enhanced enzyme immunoassay. New ultrasensitive detection systems for enzyme immunoassays, II.

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titers) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per liter. The relative intra-assay coefficients of variation of the tests were between 2 and 6%, and the interassay coefficients of variation between 4 and 12%.

Bradykinin↗
PubMed1987

A new, very sensitive, bioluminescence-enhanced detection system for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, I.

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots is described. The method utilizes antibodies conjugated with alkaline phosphatase. The alkaline phosphatase then takes part in a reaction by releasing D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light is detected by a sensitive photographic film, thereby permitting the visualization of the alkaline phosphatase-conjugated antibodies. Under non-optimized conditions the limit of detection is at present 5 to 50 pg of protein, corresponding e.g. to 30 to 300 x 10(-18) mol of rabbit immunoglobulin G. The detection system is therefore 100 times more sensitive than other systems used at present.

Alkaline Phosphatase↗
PubMed1986

Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI.

We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication. Upon transformation of this plasmid into E. coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene. Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction. Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms. Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.

Base Sequence↗
PubMed1986

Biological activity in vivo of insulin analogues modified in the N-terminal region of the B-chain.

[AlaB5]Insulin as well as a hybrid analogue of insulin and "insulin-like growth factor" (IGF-I), in which the N-terminal amino-acid sequence H-Phe-Val-Asn-Gln- of the B-chain has been replaced by the tripeptide H-Gly-Pro-Glu-of IGF-I, have been prepared by the partial-synthetic route. Their biological activity in vivo has been compared with that of other analogues in rabbits, mice and rats as far as data are available. These rodents respond differently, rats being less sensitive to modifications than rabbits and mice. The results explain unexpected discrepancies discussed in previous papers.

Amino Acid Sequence↗
PubMed1986

Granulocyte proteinases as mediators of unspecific proteolysis in inflammation: a review.

In severe inflammatory response, various blood and tissue cells, including polymorphonuclear granulocytes, release lysosomal proteinases, extracellularly and into the circulation. Such enzymes, as well as normally intracellular oxidizing agents produced during phagocytosis, enhance the inflammatory response by degrading connective tissue structures, membrane constituents and soluble proteins by proteolysis or oxidation. We first used polymorphonuclear elastase (E) as a marker of such release reactions. The liberated proteinase competes with susceptible substrates, including alpha 1-proteinase inhibitor (alpha 1PI) and alpha 2-macroglobulin, and is eliminated finally as inactive enzyme-inhibitor complexes by the reticulo-endothelial system. Using an enzyme-linked immunosorbent assay, we determined the plasma levels of E-alpha 1PI following major abdominal surgery, multiple trauma and pancreatogenic shock. Whereas the operative trauma was followed by up to 3-fold increase of the E-alpha 1-PI, postoperative septicemia was associated with a 10 to 20 fold increase. The increase of E-alpha 1-PI and a concomitant decrease of plasma factors, such as antithrombin III, clotting factor XIII and alpha 2-macroglobulin, were correlated. Multiple trauma causes a substantial increase of E-alpha 1-PI up to 14 hours after accident. The released elastase seems to correlate with severity of injury, but assessing the relationship to consumption of plasma factors is complicated by concomitant transfusions. In acute pancreatitis, peaks, of E-alpha 1-PI coincide with a massive consumption of antithrombin III and alpha 2-macroglobulin during shock.

Blood Proteins↗
PubMed1985

Tissue kallikrein of human seminal plasma is secreted by the prostate gland.

Samples of human seminal plasma were subjected to gel filtration, and the eluted fractions were analysed for their contents of tissue kallikrein-like antigen, arginine esterase activity and kininogenase activity. Two peaks of tissue kallikrein-like antigen were detected with apparent molecular masses of about 72 and 48 kDa. As judged by the criteria of molecular mass, immunoreactivity, kininogenase activity, identification of the released kinin as kallidin and inhibition studies, a genuine tissue kallikrein has been identified in the 48-kDa peak. In addition, this peak contains one or more species of immunoreactive tissue kallikrein which differ in molecular mass and enzymatic activities. The 72-kDa peak probably represents the complex of tissue kallikrein with alpha 1-proteinase inhibitor rather than a true high molecular mass tissue kallikrein. The prostate gland was identified as the site of origin of the tissue kallikrein in the seminal fluid by indirect methods and by demonstrating immunoreactive tissue kallikrein in prostatic tissue and secretion.

Chromatography, Gel↗
PubMed1985

Isolation and characterization of porcine leukocyte elastase. Leukocyte elastase-inhibitor complexes in porcine blood, II.

Porcine leukocyte elastase was purified from granulocytes by chelating chromatography on copper chelate Sepharose and by ion exchange chromatography on CM-Sepharose. Thus an enzyme preparation with a specific activity (substrate: MeOSuc(Ala)2ProValNan) of 89.3 U/mg protein was obtained. Dodecyl sulphate gel electrophoresis revealed one protein band corresponding to a molecular mass of 27 kDa. The amino acid composition was determined and isoleucine was identified as the only N-terminal amino acid residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 2000 1 . mol-1 . min-1. The dissociation constants, Ki, of the complexes of porcine leukocyte elastase with various inhibitors were calculated. The kinetic constants for the elastase-catalysed hydrolysis of MeOSuc(Ala)2ProValNan, Suc(Ala)2ValNan and Suc(Ala)3Nan were determined, as well as the kinetic constants of the inactivation of leukocyte elastase by active site mapping reagents. Detergents such as Triton X-100, Tween 20 and Brij 35, as well as porcine serum albumin, activated the porcine leukocyte elastase preparation.

Animals↗
PubMed1985

Isolation and characterization of porcine alpha 1-proteinase inhibitor. Leukocyte elastase-inhibitor complexes in porcine blood, I.

alpha 1-Proteinase inhibitor was purified from porcine blood by ammonium sulphate and Cibachron Blue-Sepharose fractionation, ion exchange chromatography on DEAE-Cellulose, gel filtration on Sephadex G-25, and zinc chelating chromatography. Thus, an inhibitor preparation with a specific activity of 1.62 IU/mg protein (enzyme: trypsin; substrate: BzArgNan) was obtained. In sodium dodecyl sulphate gel electrophoresis one protein band corresponding to a molecular mass of 67.6 kDa was found. On isoelectric focusing 6 protein bands with isoelectric points of 3.80, 3.90, 4.05, 4.20, 4.25 and 4.45 were separated. The amino acid composition was determined. The association rate constants for the inhibition of various serine proteinases were measured.

Amino Acids↗
PubMed1985

The growth of single crystal and the determination of crystallographic parameters of (L-tryptophan)A1-insulin and (D-tryptophan)A1-insulin.

The crystal-growing conditions and the results of preliminary X-ray crystallographic analysis of (L-Try)A1-insulin and (D-Try)A1-insulin are reported. The single crystals of this pair of insulin analogue suitable for X-ray diffraction analysis have been grown in the citrate buffer system by still-setting method. They both belong to the trigonal system with space group R3. The parameters of the unit cell (L-Trp)A1-insulin are aH = 80.31A, cH = 37.45A and those of (D-Trp)A1-insulin aH = 79.48A, cH = 43.81A. There are two molecules in an asymmetric unit. The obtained results are discussed.

Crystallography↗
PubMed1985

Indwelling rumino-reticulum bolus radio beacon.

A miniature battery-powered radio beacon attached to a stainless steel bolus housing an osmotic pump is described. When the bolus, of sufficient size and density to be retained in the reticulum, is administered to ruminants the radio signal can be received, using a standard AM broadcast receiver up to a range of 1.5 m. The unit allows making a simple and inexpensive verification that the drug delivery system is still within the rumino-reticulum during long-term drug delivery studies and making an external estimation of the internal anatomic location of the device.

Animals↗