Search

Das Wichtigste an der Zusammenarbeit zwischen Auftraggeber und Lieferant in einem E-Akte-Projekt ist die Haltung, ein gemeinsames Team fur ein gemeinsames Ziel darzustellen. Dann konnen die vielfaltigen Anderungen, die der Wirbelwind des Tagesgeschafts erzwingt, in einem Geist der Kooperation gemeinsam vereinbart werden. Nicht starres Festhalten an vordefinierten Leistungskatalogen, sondern die standige Optimierung des Projektnutzens ist der Garant des Erfolgs. Mit Scrum haben wir auf diesem Wege – der „Maximierung nicht getaner Arbeit“ – gute Erfahrungen gemacht.

With a new immunoenzymometric assay we measured human glycogen phosphorylase isoenzyme BB (GPBB) in 116 healthy individuals, 14 patients with stable angina, 107 nontraumatic chest pain patients on admission to the emergency department [45 acute myocardial infarction (AMI), 49 unstable angina, 13 other diseases], and in serial samples from 41 AMI patients. GPBB was compared with creatine kinase (CK), CKMB mass, myoglobin, and cardiac troponin T. Receiver-operating characteristic plots demonstrated the significantly greater (P < or = 0.012) discriminatory power of GPBB to detect acute ischemic coronary syndromes compared with all other tested markers. GPBB was the most sensitive marker for detection of AMI during the first 4 h after onset of chest pain, and only GPBB was increased above the upper reference limit (7 micrograms/L) on admission in patients who had unstable angina at rest and reversible ST-T alterations. This and the high early sensitivity of GPBB are most likely explained by its function as a key enzyme of glycogenolysis.

A highly specific, quick and sensitive immunenzymometric assay (IEMA) was developed for human interferon-alpha-1 (HuIFN-alpha-1) and human interferon-alpha-2 (HuIFN-alpha-2), respectively. Due to the specificity of the used antibodies it is possible to measure HuIFN-alpha-1 and HuIFN-alpha-2 also in the presence of the corresponding other interferon (IFN) in body fluids as serum or plasma and in culture supernatants. These two-site binding assays are constructed for HuIFN-alpha-1 form tow monoclonal antibodies (ZIM-VD11 and ZIM-IIG11) and for HuIFN-alpha-2 from a sheep antibody against leukocyte-IFN and a monoclonal antibody (ZIM-IIG7). One incubation step is necessary only: for HuLFN-alpha-1, 90 minutes (min), for the determination of HuIFN-alpha-2, 150 min. Very low detection limits characterize these IEMAs: for HuIFN-alpha-1: 0.5 IU/ml, and for HuIFN-alpha-2: 20 IU/ml. Both assays detect the corresponding IFNs as natural and recombinant molecules as well. Only biologically active IFN molecules can be m...

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC. It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steffen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.), 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a chromatographic run, was avoided by adding sodium chloride to both separation buffers.

The diagnosis of myocardial damage is preferably based on measurement of the cardiac-specific troponins. However, there is an emerging need for early, specific cardiac markers. One potential candidate is the glycogen phosphorylase BB isoenzyme (GPBB). We investigated the use of a new, commercially available GPBB ELISA assay in 61 patients presenting with an acute coronary syndrome (37 acute myocardial infarction, 24 unstable angina pectoris) in comparison to established cardiac markers such as troponin T, creatine kinase isoenzyme MB (CKMB) mass, and myoglobin. Blood samples were obtained on arrival, as well as 1, 2, 3, 4, 8, 12 and 24h later. GPBB plasma concentrations were elevated in 90.9% of patients 1h after onset of chest pain and increased to 100% at 4–5h. Within the first 6h, GPBB showed the highest sensitivity (95.5–100%) and high specificity (94–96%) compared to myoglobin (85–95% sensitivity) and CKMB mass (71.4–91.3% sensitivity). As expected, troponin T showed high specificity (100%) and sensitivity >95% later in the time course (≥3h). In un-stable angina pectoris patients, a very high rate of elevated GPBB was observed (93.9% at 3h) compared to myoglobin (66.7%). Cardiac troponin T and CKMB were only elevated in 33.8% and 55.0% of these patients, respectively. In conclusion, GPBB is a promising marker for the early diagnosis of acute coronary syndromes and could probably act as a marker of ischemia. However, further studies on specificity and development of a fast, automated assay are necessary before GPBB can be recommended as a routine diagnostic tool.

As with any new test, novel cardiac markers must meet basic analytical and clinical performance standards. As far as sensitivity and specificity are concerned, it is important to know the nature of the proteins immediately released into the blood after cardiac damage. Early identification and confirmation of acute myocardial injury is essential for appropriate patient care and management in the emergency department. In this respect, glycogen phosphorylase (GP) is a sensitive, early released, and suitable cardiac marker protein. This enzyme plays an essential role in the anaerobic energy metabolism during myocardial oxygen deficiency. In the aerobic heart muscle, GP and glycogen are closely associated with the vesicles of the sarcoplasmic reticulum. In tissue, hypoxic glycogen degradation is catalyzed by the GP isoenzyme BB, which is the main isoform in the human myocardium. Ischemia favours the temporary conversion of GPBB into the phosphorylated a form thereby accelerating glycogen breakdown. Within minutes the cytosolic GPBB is dephosphorylated to the b form which is then free to circulate in the cytoplasm. GPBB can diffuse out of the cell if cell membrane permeability is also increased, as is usually the case in hypoxia. This sequence reflects a unique structural and functional interaction between GPBB and the ischemic-sensitive process of glycogenolysis and membrane alteration. This concept is novel and differs from that of other markers of myocardial damage, such as myoglobin, creatine kinase(s), and troponins, as well as fatty-acid-binding proteins, when the efflux reflects alterations in cell membrane integrity.

OBJECTIVE--To determine whether transient ST-T alterations in patients with unstable angina are associated with an increase in plasma glycogen phosphorylase BB concentrations on admission to hospital. DESIGN--Prospective screening of patients with unstable angina for markers of myocardial cell damage. SETTING--Accident and emergency department of university hospital. PATIENTS--48 consecutive patients admitted for angina pectoris (18 with transient ST-T alterations). None of the patients had acute myocardial infarction according to standard criteria. MAIN OUTCOME MEASURES--Creatine kinase and creatine kinase MB activities, creatine kinase MB mass concentration, and myoglobin, cardiac troponin T, and glycogen phosphorylase BB concentrations on admission. RESULTS--All variables except for creatine kinase and creatine kinase MB activities were significantly higher on admission in patients with unstable angina and transient ST-T alterations than in patients without. However, glycogen phosphorylase BB concentration was the only marker that was significantly (p = 0.0001) increased above its discriminator value in most patients (16). In the 18 patients with transient ST-T alterations creatine kinase MB mass concentration and troponin T and myoglobin concentrations were significantly (p = 0.0001) less commonly increased on admission (in five, three, and two patients, respectively). CONCLUSIONS--The early release of glycogen phosphorylase BB may help to identify high risk patients with unstable angina even on admission to an emergency department. Glycogen phosphorylase BB concentrations could help to guide decisions about patient management.

This review deals with glycogen phosphorylase (GP) and its isoenzyme BB in the diagnosis of ischaemic myocardial injury. Early identification and confirmation of acute myocardial infarction is essential for correct patient care and disposition decision in the emergency department. In this respect, glycogen phosphorylase isoenzyme BB (GPBB) based on its metabolic function is an enzyme for early laboratory detection of ischaemia. In the aerobic heart muscle GPBB together with glycogen is tightly associated with the vesicles of the sarcoplasmic reticulum. Release of GPBB, the main isoform in the human myocardium, essentially depends on the degradation of glycogen, which is catalyzed by GP. Ischaemia is known to favour the conversion of bound GP in the b form into GP a, thereby accelerating glycogen breakdown, which is the ultimate prerequisite for getting GP into a soluble form being able to move freely in the cytosol. The efflux of GPBB into the extracellular fluid follows if ischaemia-induced structural alterations in the cell membrane become manifest. The clinical application of GPBB as a marker of ischaemic myocardial injury is a very promising tool for extending our knowledge of the severity of myocardial ischaemic events in the various coronary syndromes. The rational roots of this development were originated from Albert Wollenberger's research work on the biochemistry of cardiac ischaemia and the transient acceleration of glycogenolysis mainly brought about by GP activation.

Novel kinds of magnetizable particles have been prepared using the interaction between the complexing groups of phosphonic acid and polyvalent metal ions on the surface of Fe3O4 particles. After modification of monoclonal and polyclonal antibodies by the bifunctional chelating agent 2-(4-di-azophenyl)-1-hydroxyethane-1,1-bisphosphonic acid, antibodies were immobilized at a ratio of 5–10 mg antibody molecules per ml Fe3O4 particles without loss of immunological reactivity. Very sensitive and fast immunoenzymometric assays for the quantitative determination of human interferon-α1 and mouse immunoglobulins were developed using such particles. The advantages of the method include immobilization of proteins on magnetizable carriers without chemical modification of the carrier and the short assay time compared to conventional immunoenzymometric assays.

SummaryThe study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding.Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D’). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA.The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F. Weak inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic activity is suppressed only by B-chain specific antibody 22.

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred micrograms of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.

Leberextrakt hat in Gegenwart von L-Glutamat eine hemmende Wirkung auf die anaerobe Glykolyse der Ehrlich-Mäuseascites-Carcinomzelle. Die hemmende Wirkung des Leberextraktes wird auf die Wirkung von Glutamat-Pyruvat-Transaminase zurückgeführt. Das bei der Glykolyse intermediär entstehende Pyruvat wird durch die Transaminierungs-Reaktion abgefangen und somit der Dehydrierung von DPNH entzogen. Die sinkende Konzentration an DPN ⨁ hat eine Hemmung der Triosephosphatdehydrierung zur Folge. Die Glykolysehemmung durch GPT ist aufhebbar durch Sauerstoff sowie durch Zugabe von Pyruvat, von DPN ⨁, von α-Ketoglutarat und von L-Alanin.

Es wird ein Verfahren zur Darstellung hoch gereinigter Glutamat-Pyruvat-Transaminase beschrieben. Die anaerobe Glykolyse in Ehrlich-Mäuseascites-Carcinomzellen wird durch hoch gereinigte GPT ebenso gehemmt wie durch Leberextrakt, wenn die gleiche Konzentration an GPT-Einheiten eingesetzt wird.

Nucleolar ‘80-S’ and ‘40-S’ preribosomes (containining 45-S and 21-S pre-rRNA, respectively), as well as cytoplasmic ribosomes, were isolated from Friend erythroleukemia cells. The presence of structural ribosomal proteins in the isolated particles was studied by using antisera against individual rat liver small ribosomal subunit proteins. The analysis is based on the established crossreactivity between rat and mouse ribosomes [F. Noll and H. Bielka (1970) Mol. Gen. Genet. 106, 106–113]. The identification of the proteins was achieved by two independent immunological techniques: the passive haemagglutination test and the enzyme immunoassay of electrophoretically fractionated proteins, blotted on nitrocellulose. All 17 proteins tested are present in cytoplasmic ribosomes. A large number of proteins (S3a, S6, S7, S8, S11, S13, S14, S18, S20, S23/24 and S25) are present in the ‘80-S’ pre-ribosome. Only two proteins (S3 and S21) are added during the formation of the ‘40-S’ preribosome in the nucleolus. Four proteins (S2, S19, S26 and S29) are added at later, possibly extranucleolar, stages of ribosome formation. The results obtained provide evidence for the sequential addition of proteins during the formation of the small ribosomal subunit in Friend erythroleukemia cells.