Irene Witt

Interleukin-6 and α-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices

Recent studies indicated that the formation of a major constituent of Alzheimers disease (AD) senile plaques, called βA4‐peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its βA4 sequence was found to be part of APP processing the formation of the βA4‐peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, α‐2‐macroglobulin (α2M), upon stimulation with the inflammatory mediator interleukin‐6 (IL‐6) we now investigated whether α2M and IL‐6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong α2M and IL‐6 immunoreactivity while no such immunoreactivity was found in age‐matched control brains. Strong perinuclear α2M immunoreactivity in hippocampal CAI neurons of Alzheimers disease brains indicates that neuronal cells are the site of α2M synthesis in AD brains. We did not detect elevated IL‐6 or α2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology.

Isoenzyme der malatdehydrogenase und ihre regulation in Saccharomyces cerevisiae

Abstract 1. 1. From Saccharomyces cerevisiae , incubated on a glucose-free medium with acetate as the only carbon source, two different malate dehydrogenases ( l -malate: NAD + oxidoreductase, EC 1.1.1.37) have been isolated by DEAE-cellulose ion-exchange chromatography. One of these enzymes was only found in the mitochondria and is called enzyme A or m-malate dehydrogenase; the other enzyme was found in the extramitochondrial c-space and is called enzyme B or c-malate dehydrogenase. At present it cannot be decided whether m-malate dehydrogenase also exists in the c-space or leaks when the mitochondria are injured. 2. 2. The reaction velocity plotted against the concentration of oxaloacetic acid showed a characteristic substrate inhibition in the case of m-malate dehydrogenase In contrast, c-malate dehydrogenase showed no substrate inhibition. This difference corresponds to the behaviour of m-malate dehydrogenase and c-malate dehydrogenase from liver. 3. 3. In yeast grown on glucose only m-malate dehydrogenase could be found, but after incubating the cells on acetate as the sole carbon source, both m-malate dehydrogenase and c-malate dehydrogenase were found. In reference to earlier experiments concerning the regulation of malate dehydrogenase activity in yeast, it is concluded that a repression of c-malate dehydrogenase synthesis by glucose occurs. This regulating mechanism is useful for the cell, because in the glycoxylate cycle c-malate dehydrogenase participates in the gluconeogenesis from acetate or ethanol. This enzyme is not necessary when glucose is in the medium.

Increased phosphorylation of human fibrinopeptide A under acute phase conditions

The High Performance Liquid Chromatography (HPLC) separation of the fibrinopeptides liberated by the action of thrombin from plasma fibrinogen in a new one-step procedure without prior purification of fibrinogen is described. Since the phosphorylated and non-phosphorylated form of fibrinopeptide A are clearly resolved by this method, the determination of the degree of phosphorylation of fibrinopeptide A from the peak heights of these peptides becomes possible. By this method the degree of phosphorylation of fibrinogen in healthy volunteers (n = 21) is found to be 23.6 +/- 3.6%. Under acute phase conditions where the synthesis rate of fibrinogen is known to be markedly enhanced the degree of its phosphorylation also increases considerably. This was demonstrated on 13 patients undergoing an elective hip joint replacement, the hip surgery being chosen as a model for the elicitation of an acute phase reaction. The degree of phosphorylation rises steeply up to 60% on the first day after operation thereafter declining slowly to normal values within about one week. The maximum of the degree of phosphorylation precedes that of the fibrinogen concentration by several days. The mechanism which leads to the higher phosphorylation of fibrinogen during increased synthesis is unknown at the moment.

Influence of organically bound phosphorus in foetal and adult fibrinogen on the kinetics of the interaction between thrombin and fibrinogen

Abstract Fibrinogen contains a phosphoserine residue replacing serine at position 3 from the N-terminal end of the α(A)-chain. Foetal fibrinogen was found to contain twice as much organically bound phosphorus as adult fibrinogen. Whether the properties of fibrinogen are influenced by the phosphorus moiety was studied on dephosphorylated fibrinogen samples. Dephosphorylation does not alter the conversion rate of foetal or adult fibrinogen to fibrin. Neither is the affinity between fibrinogen and thrombin influenced by the phosphorus content.

The location of a second in vivo phosphorylation site in the Aα-chain of human fibrinogen

Abstract By quantitative phosphorus determination on the single chains of human fibrinogen it is demonstrated that the covalently bound phosphorus of adult and fetal fibrinogen is exclusively located in the Aα-chain. The Aα-chain of fetal fibrinogen contains about twice as much phosphorus as the adult Aα-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the Aα-chain. By consecutive cleavage of the Aα-chains of fetal and adult fibrinogen with cyanogen bromide, trypsin, and chymotrypsin, separation of the resulting peptide mixtures and analysis for phosphorylated amino acids, this second phosphorylation site could be traced to Ser 345 of the Aα-chain. There is only one sequence homology between the two now known in vivo phosphorylation sites of human fibrinogen, namely that the second amino acid to the carboxyl side of the phosphorylated Ser is Glu. The sequence specificity of the up to now unidentified protein kinase phosphorylating fibrinogen allows it to be classified as a member of the group of type-2 casein kinases or casein kinases TS.

Vergleichende biochemische Untersuchungen an Erythrocyten aus Neugeborenen-und Erwachsenen-Blut

ZusammenfassungDie Aktivitäten der untersuchten Enzyme der Glykolyse, des Citronensäurecyclus, des Pentosephosphatcyclus sowie der glutathionreduzierenden Enzyme sind in Erythrocyten aus Neugeborenenblut zwischen 1,3–3,0 mal so hoch wie in Erythrocyten aus Erwachsenenblut, bis auf eine Ausnahme: 6-Phosphofructokinase ist in beiden Zelltypen gleich aktiv. Die Glykolyserate ist in Neugeborenenerythrocyten etwa 40% höher als in Erwachsenenerythrocyten. Die Konzentrationen von ATP, ADP und NAD sind in beiden Zelltypen gleich, während das anorganische Phosphat in Neugeborenenzellen um 40% höher ist als in Erwachsenenzellen.Die Ergebnisse lassen keine Entscheidung darüber zu, ob die Besonderheiten der Neugeborenenerythrocyten durch das Vorliegen einer besonderen Zellpopulation während der Neugeborenenperiode oder den hohen Gehalt an jungen Zellen im Neugeborenenblut zustande kommen.SummaryThe investigated enzymes of glycolysis, pentose phosphate cycle, citric acid cycle and the glutathione reducing enzymes have 1,3–3,0 times higher activities in erythrocytes from blood of newborns than in erythrocytes from blood of adults. In contrast to the other enzymes of glycolysis 6-phosphofructokinase has the same activity in both types of cells. The rate of glycolysis is 40% higher in erythrocytes from newborns than in erythrocytes of adults.The concentrations of ATP, ADP and NAD are the same in both types of cells, whereas the concentration of inorganic phosphate is 40% higher in erythrocytes from newborns. The results do not allow a decision, if the peculinarities in the erythrocytes of newborns are due to a special erythrocyte population during the newborn-period or to the higher content of young erythrocytes in the blood of newborns.

Regulation of pyruvate decarboxylase (E.C.4.1.1.1) synthesis by coenzyme induction in Saccharomyces cerevisiae

Abstract Aerobic glycolysis increases rapidly after the addition of ammonia to yeast cells which are oxidizing glucose. This increase in glycolytic rate is caused by ammonia dependent ATP-consuming synthetic reactions and the subsequent increase of the levels of orthophosphate and ADP, which are rate-limiting for glycolysis ( Holzer, 1953 ; Holzer and Witt, 1958 ; Witt and Holzer, 1964 ). This rapid regulation of glycolysis depends on the activation or inhibition of glycolytic enzymes by metabolites or coenzymes. In this paper we present results for a slower glycolytic control mechanism involving the regulation of the synthesis of a rate-limiting enzyme. Incubation of starved yeast cells with glucose and a nitrogen source led to a 2 – 4 fold increase of the pyruvate decarboxylase (PD) activity and the thiamine content within 3 hours. The activity of PD could also be increased by addition of thiamine to the medium. Therefore we assume that the PD activity may be regulated by the thiamine content of the cells. This assumption was supported by the observation that in thiamine-deficient mutants of Saccharomyces cerevisiae the rate of PD synthesis depended on the thiamine content of the growth medium.

N-terminal amino acid sequences of precursor and mature forms of α-1-antitrypsin

α‐1‐Antitrypsin is found in hepatocytes as a high‐mannose glycoprotein (M r 49000), extracellularly as a complex‐type glycoprotein (M r 54000). Deglycosylation of both forms with peptide: N‐glycosidase led to proteins of identical app. M r (41000). The sequence of 26 N‐terminal amino acids of rat α1‐antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized α1‐antitrypsin showed a cluster of hydrophobic amino acids. A pre‐peptide of 24 amino acids is proposed. There is no evidence for the existence of a propeptide.

Increase in the degree of phosphorylation of circulating fibrinogen under thrombolytic therapy with urokinase

Human fibrinogen is phosphorylated in vivo to an equal extent at two positions, one at Ser 3 located on fibrinopeptide A, the other at Ser 345 of the A alpha-chain. As has been shown previously, the degree of phosphorylation of the circulating fibrinogen pool can be determined in vitro from the ratio between the HPLC peaks formed by phosphorylated and non-phosphorylated fibrinopeptide A which has been cleaved from plasma fibrinogen by thrombin or reptilase. Plasma samples were obtained from patients with venous thrombosis undergoing fibrinolytic therapy with urokinase (n = 8). The degree of phosphorylation increased from about 35% before treatment to values between 50% and 70% within 48 hours. It remained at these high levels as long as urokinase was administered and declined slowly thereafter. This behaviour of the degree of phosphorylation of fibrinogen is explained by a model which assumes that fibrinogen is secreted in the phosphorylated form and then dephosphorylated in the circulation by an up to now unidentified phosphatase by first order kinetics. When this system is in steady state, the degree of phosphorylation is about 25% under normal conditions. If the elimination rate of fibrinogen is greatly enhanced by fibrinogenolysis the system will approach a new steady state with a higher degree of phosphorylation, the magnitude of which will depend on the new ratio of dephosphorylation and elimination.

Electron microscopic studies on the foetal fibrin clot

Abstract Morphometric analyses of electron microscopic pictures revealed that the fibre segments of foetal fibrin are nearly half as thick and long as those of adult fibrin. The network of foetal fibrin is more compact, with a density of 49 % as compared to adult fibrin with 31 %. These results may explain earlier observations that the fibrin clots from the blood of newborns are more transparent and exhibit lower compressibility than those from adult blood.