Characterisation of calmodulin from Drosophila heads.

Calmodulin from Drosophila heads has been purified to apparent electrophoretic homogeneity. It has the same characteristics as bovine brain calmodulin with respect to the migration upon polyacrylamide gel electrophoresis and maximal activation of a calmodulin-deficient cAMP phosphodiesterase. The amino acid composition resembles bovine brain calmodulin with the exception that trimethyllysine is absent and that it contains only one tyrosine. The tryptic peptide map of Drosophila calmodulin suggests some differences in the amino acid sequence as compared to bovine brain calmodulin. These proposed differences in the primary structure may explain why Drosophila calmodulin is less potent than bovine brain calmodulin in the activation of a cAMP phosphodiesterase from bovine brain.

Pattern of thyrotropin and thyroxine plasma concentrations during the 24-hour sleep-wake cycle in the male rhesus monkey.

The 24-h pattern of the plasma TSH concentration was investigated in five male rhesus monkeys prepared chronically with right atrial catheters and electroencephalogram (EEG) electrodes. The preparation allowed frequent blood sampling (every 15 min), TV monitoring, and EEG recording from the adjacent room for extended periods of time from undisturbed animals. In addition, nap deprivation, 5 h total sleep deprivation, and specific sleep stage deprivation experiments were performed in order to test their influence on the TSH pattern. T4 was also determined in approximately half of the profiles during undisturbed conditions. Both TSH and T4 patterns consisted of low amplitude, high frequency fluctuations which, however, did not exceed the assay variation. TSH showed superimposed higher amplitude spikes at unpredictable times (0-5/24-h). Intra- and interanimal variabilities of both TSH and T4 patterns were high. Power spectral maxima of the TSH time series indicated periodicities between 30-75 min which were not significant. No nyctohemeral difference in the TSH or T4 pattern or their mean concentrations was found, and there was no consistent pattern of a circadian cycle. Independent changes of average TSH and T4 concentrations were seen in 5 of the 16 profiles during undisturbed conditions, under which both hormones were determined. Cross-correlation analysis of the hormonal time series revealed no significant relation between TSH and T4 patterns. The deprivation procedures had no significant influence on the day or nighttime pattern of TSH. Cross-correlation analysis showed no relation between TSH and either activity during the day or sleep stages during the night. It is concluded that in the rhesus monkey, in contrast to man, TSH secretion shows no circadian variation and is not influenced by the sleep-wake cycle.

Twenty-four-hour pattern of growth hormone secretion in the rhesus monkey: studies including alterations of the sleep/wake and sleep stage cycles.

The 24-h pattern of GH secretion and its possible relation to the sleep/wake cycle and to sleep stages were studied in 12 male rhesus monkeys. Blood samples were drawn every 15 min for 96 h, 24 h, or shorter periods of time through chronic right atrial catheters which extended through the wall into the adjacent room. In addition, activity rating (daytime) and determination of sleep stages from electroencephalogram recordings (nighttime) were done. GH profiles were obtained during undisturbed conditions and during deprivation of nap, 5 h total sleep, slow wave sleep (SWS), and rapid eye movement (REM) sleep. GH secretion was episodic, with peak concentrations often exceeding 20 ngeq/ml and nadirs mostly below 1 ngeq/ml. Autocorrelation analysis demonstrated a circadian and an ultradian rhythm during undisturbed conditions. However, the cycle length of the ultradian rhythm showed large inter- and intraindividual variations (from 3--6 h). Neither cross-correlation analysis between hormonal and activity/electroencephalogram sleep stage time series nor results of deprivation experiments produced evidence for a link between nap phases, the sleep/wake cycle, or the SWS/REM sleep stage cycle on the one hand and the GH secretory pattern on the other hand. However, while SWS deprivation was highly effective, REM deprivation did not substantially reduce total REM sleep time due to frequent entries into abortive REM sleep epochs. During the daytime, there was no significant correlation between activity/arousal and GH, but during the night, there was a significant positive correlation between stage waking and GH. A direct or indirect synchronizing effect of the matutinal light change is suggested by the pattern of the 24-h curve of mean GH concentrations during undisturbed conditions: a steep increase from very low concentrations at light onset, followed by a succession of nadirs and peaks at approximately 4.5-h intervals. However, the nadirs became progressively more shallow until there was no apparent periodicity during the night due to the loss of synchronization. It is concluded that GH in the rhesus monkey shows a circadian and an ultradian periodicity. However, in contrast to man, sleep and SWS are not important determinators of the 24-h GH pattern.

Two genes encoding ribosomal protein L3 of Schizosaccharomyces pombe and their proximal promoter regions.

We have cloned and sequenced two genes, rpl3-1 and rpl3-2, encoding the ribosomal protein L3 of Schizosaccharomyces pombe. The two genes contain an open reading frame encoding 388 amino acids (aa) with a M(r) of 43,808. The aa sequences are identical, except at position 78, where Rpl3-1 displays a valine residue and Rpl3-2 contains isoleucine. The aa sequences show 75% identity to the RPL3 aa sequence from Saccharomyces cerevisiae. S1-nuclease protection analysis revealed that both genes are transcribed. The promoter sequences of the two rpl3 genes are significantly different, but both promoters contain the conserved homol-D element. Transcription starts between 40 and 50 nt downstream from this element.

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

alpha-1-Antitrypsin is found in hepatocytes as a high-mannose glycoprotein (Mr 49 000), extracellularly as a complex-type glycoprotein (Mr 54 000). Deglycosylation of both forms with peptide: N-glycosidase led to proteins of identical app. Mr (41 000). The sequence of 26 N-terminal amino acids of rat alpha 1-antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized alpha 1-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.

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

Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-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, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M 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.

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.

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.

The location of a second in vivo phosphorylation site in the A alpha-chain of human fibrinogen.

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 A alpha chain. The A alpha-chain of fetal fibrinogen contains about twice as much phosphorus as the adult A alpha-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the A alpha-chain. By consecutive cleavage of the A alpha-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 A alpha-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.

A novel homozygous missense mutation (Val 325-->Ala) in the protein C gene causing neonatal purpura fulminans.

A novel homozygous GTG-->GCG (Val 325-->Ala) substitution was detected in the protein C gene of a newborn causing severe purpura fulminans post partum. In the consanguineous parents and two further infants a heterozygous type 1 protein C deficiency was found. Up to now the heterozygous individuals are clinically unaffected. The mutation co-segregates with the protein C deficiency state. It creates a restriction enzyme (Sac II) cleavage site.

Fibrinogen Kiel: a congenital dysfibrinogenaemia with (A alpha-16 Arg----His) substitution characterized by HPLC without prior isolation of fibrinogen.

A congenital fibrinogen variant in a German family is described which has been identified as a substitution of His in position 16 of the A alpha-chain for Arg, manifested over three generations in heterozygous form. The characterization is based on the reaction of the variant fibrinogen with thrombin and reptilase, on the HPLC-chromatographic properties and the amino acid composition of the abnormal fibrinopeptide A. Clinical observations in the affected family members (neither haemorrhagic nor thrombophilic tendencies), the results of routine coagulation tests (normal global clotting tests, prolonged thrombin and thrombin-coagulase time, decreased fibrinogen concentration in functional as opposed to immunological tests), and the autosomal co-dominant modus of inheritance of the fibrinogen variant are all in complete agreement with other reports in the literature concerning the same amino acid exchange. The results of our experiments with fibrinogen Kiel allow no definite conclusion regarding the question of whether it consists of pure homodimers or as of a mixture of homo- and heterodimers.

Rapid identification of gene defects in protein C deficiency by temperature gradient gel electrophoresis.

Protein C deficiency is an autosomally inherited disorder that is associated with a high risk of recurrent venous thrombosis. The authors have shown that temperature gradient gel electrophoresis (TGGE) is a simple and rapid screening method for the detection of mutations in the protein C gene. Samples from eleven patients with sequence defined point mutations in the promoter region of exon I, and in exons II, III, VII, VIII and IX were analysed by TGGE. In all cases the mutations were readily detected. The exons IV, V and VI were not submissive to TGGE analysis due to amplification difficulties. However, specific computer calculations predict a more general applicability of TGGE for the detection of any mutation in the protein C gene. The presented data establish the usefulness of TGGE as a simple and rapid screening method for the detection of hereditary mutations in the protein C gene.

The risk of developing breast cancer in relation to mammography findings.

Several studies have shown that it is possible to use the parenchymal patterns disclosed by mammography to differentiate various groups with a high risk of developing breast cancer. Accordingly, a patient series of 597 women who had undergone mammography during the period 1971-1975 was examined and classified according to Wolfe's system. A follow-up of 513 patients five to nine years after the mammography revealed no statistical difference in the tendency to develop breast cancer using Wolfe's parenchymal pattern method. The study shows that those in the group with pronounced fibroadenomatous changes, which were primarily studies radiologically, have an incidence of breast cancer three times as high as that found in the population as a whole.

A comparative analysis of x-ray findings of the lumbar spine in patients with and without lumbar pain.

Roentgenograms of the lumbar spine from 238 patients with lower back pain (LBP) with sciatica were compared with roentgenograms from 66 patients without LBP. No difference between the two groups could be demonstrated concerning the incidence of spondylosis and disc degeneration. Cases with wedge-shaped vertebral bodies are significantly more frequent in patients younger than 40 years old in the group having pain. The incidence of spondylosis and disc degeneration increase with increasing age.

[Molecular biological basis and diagnosis of hereditary defect of antithrombin III, protein c and protein S]. / Molekularbiologische Grundlagen und Diagnostik der hereditären Defekte von Antithrombin III, Protein C und Protein S.

Recent progress in molecular biology enabled the elucidation of the nucleotide sequences of the genes for antithrombin III (AT III), protein C (PROC) and protein S (PROS). Furthermore numerous mutations were identified causing genetic defects of the important inhibitors of blood coagulation. As the genes for AT III (13.8 kb) and PROC (11.2 kb) are small and easy to analyze a great number of molecular defects already are described in extensive databases (50, 73): 79 different mutations for AT III and 160 for PROC are included. The identification of mutations leading to AT III and PROC deficiency has given important information on the structure-function relationships of the proteins. In case of protein C deficiency the clinical relevance of DNA analyses is most important because the diagnosis at the protein level is often uncertain. The gene for PROS is not so easy to analyze like the other two genes. The PROS gene is large and also a PROS pseudogene exists. Although a number of mutations have been identified, there has not been published a database until now. The clinical relevance to identify gene defects in PROS deficiency is as important as for PROC deficiency. Presumably the elucidation of PROS gene defects will advance in the near future.