Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction.

An improved method for the determination of ethyl glucuronide (EtG) in human serum and urine was developed using solid-phase extraction (SPE) and gas chromatography (GC) with mass spectrometric detection (MS). EtG was isolated from serum and urine using aminopropyl SPE columns after deproteination with perchloric acid and hydrochloric acid, respectively. The chromatographic separation was performed on a DB 1701 fused-silica column. At a signal-to-noise ratio of 3:1, a quantification limit of 173 and 560 ng/ml and a detection limit of 37 and 168 ng/ml could be determined for serum and urine, respectively. This indicates high specificity and sensitivity of the described method. The mean absolute recovery was approximately 85%, while intra- and inter-day precision of the assay were all less than 7.5%. The linearity of the calibration curves was satisfying as indicated by correlation coefficients of >0.993. The presented method provides the basis for determination and identification of EtG in human serum and urine samples in a low-concentration range for monitoring alcohol consumption during treatment for alcohol dependence and comorbid alcohol abuse of psychotherapy patients.

Cerebral microembolism, a disease marker for ischemic cerebrovascular events in the antiphospholipid syndrome of systemic lupus erythematosus?

OBJECTIVES: We investigated whether the detectability of microembolic Doppler signals (MES) in the intracranial circulation may help to define the individual cerebrovascular risk in systemic lupus erythematosus (SLE) with antiphospholipid syndrome (APS). MATERIAL AND METHODS: Retrospective cross-sectional study of 70 patients with SLE with or without APS, and 30 controls with a history of cerebral ischemia of unknown cause. Of all patients, 38 had a clinical history of APS and 32 did not. RESULTS: 15 patients with APS (39%) showed MES. In contrast, all patients without APS and 29 of 30 controls were microemboli-negative. MES were more strongly associated with cerebrovascular symptoms than with APS, antiphospholipid antibodies, or cardiac pathology. The time elapsed since the last ischemic cerebrovascular symptom was significantly shorter in microemboli-positive patients than in microemboli-negative patients (P<0.001). CONCLUSION: MES may be related to disease activity in patients with SLE and APS. Their detection may help to assess individual cerebrovascular risk and contribute to therapeutic decision making and therapeutic monitoring.

Protein kinase associated with ribosomes phosphorylates ribosomal proteins of Streptomyces collinus.

Protein kinase activity associated with ribosomes of a kirromycin-producing strain of Streptomyces collinus was detected. The enzyme utilizes [gamma-32P]ATP to phosphorylate proteins, yielding acid-stable phosphoamino acids. Two-dimensional electrophoresis of proteins from a crude ribosomal fraction revealed 17 phosphoproteins. Eleven of the phosphoproteins exhibited electrophoretic mobility identical to that of S. collinus ribosomal proteins S3, S4, S12, S13, S14, S18, L2, L7, L16, L17, and L23. Protein L2 was identified by microsequencing of internal peptide fragments. Immunodetection with monoclonal antibodies indicated that the ribosomal proteins are phosphorylated on serine and threonine residues. Phosphorylation of ribosomal proteins led to the reduction of activity of ribosomes in the translation of poly(U). These results provide the first evidence of phosphorylation of ribosomal proteins in bacteriophage-uninfected cells of eubacteria.

Cerebral microemboli in patients with antiphospholipid syndrome.

CNS involvement is one of the hallmarks underlying poor prognosis in SLE and for therapeutic strategies it is difficult to assess which patients are at risk. As detectability of cerebral microembolic signals (MES) using long-term transcranial Doppler ultrasonography (TCD) proved to be predictive of past and future thromboembolic events in patients with carotid artery stenosis, we investigated whether MES might be similarly useful in patients with antiphospholipid syndrome (APS). Our study population consisted of 46 SLE patients and five with primary APS (pAPS). Twelve of the 46 patients with SLE, 10 of them with secondary APS (sAPS), and four of five patients with pAPS had a history of cerebrovascular ischaemia (CVI). MES in the middle cerebral artery were detected in 14 of 16 patients with and in one of 35 without CVI (P < 0.001). Antiphospholipid antibodies (aPL) were positive in 12 of 15 patients with and 18 of 36 without MES, and the rate of MES correlated to the titre of aPL. MES from APS patients resembled in their energy distribution those from patients with symptomatic carotid disease and were significantly different from those associated with artificial heart valves. In conclusion, cerebral MES are detectable in APS patients and correlate with a history of CVI. Whether this innovative method may provide individual risk assessment in APS patients has to be addressed in prospective studies.

[Thallium perfusion scintigraphy and bicycle ergometry in the diagnosis of ischemic heart disease. Comparison with coronarography findings]. / Perfuzní thaliová scintigrafie a bicyklová ergometrie v diagnostice ischemické choroby srdecní. Srovnání s koronarografickými nálezy.

In 76 patients (66 men and 10 women, age 20-71 years) with stable angina pectoris planar thallium myocardial perfusion scintigraphy, bicycle ergometry and coronary angiography were performed. Thallium scintigraphy was highly sensitive (97%) for detection of ischaemic heart disease, the sensitivity of ergometry was 75%. When comparing patients with affected 1, 2 and 3 coronary arteries perfusion scintigraphy had a sensitivity of 100%, 90% and 100% resp., while bicycle ergometry 62%, 90% and 82% resp. The specificity of perfusion scintigraphy was low (47%), compared with ergometry (60%). The finding of s reversible perfusion defect during scintigraphy indicated significant affection of coronary arteries. The scintigraphic finding of diffuse myocardial ischaemic affection was not significant for assessment of the severity of the coronary artery disease.

Cerebral microembolism in patients with Sneddon's syndrome.

BACKGROUND: The pathogenesis of Sneddon's syndrome is unclear. This study addresses the question whether cerebral thromboembolism may be involved in the pathogenesis of the neurologic complications of the disorder. The study consisted of 13 patients with Sneddon's syndrome defined by both generalized livedo reticularis and a history of one or more cerebrovascular ischemic events; none had clinical or Doppler ultrasonographic evidence of atherosclerosis. METHODS: Transcranial Doppler microembolic monitoring of the middle cerebral artery; blood screening for antiphospholipid antibodies (lupus anticoagulant, anticardiolipin antibodies). RESULTS: Five patients (38%) showed clinically silent microembolism at transcranial Doppler monitoring, with individual microembolic event rates of the middle cerebral artery between 2 per hour and 33 per hour. In this group, the time since the last ischemic symptom was significantly shorter than in the eight patients without microemboli. Antiphospholipid antibodies were detected in three patients (23%), all of whom belonged to the microemboli-positive group. CONCLUSIONS: These data suggest that the detectability of both clinically silent cerebral microembolism and antiphospholipid antibodies may provide paraclinical evidence of active disease in patients with Sneddon's syndrome. The results support the notion that an immune-mediated prothrombotic state facilitating the formation of arterial thrombi with subsequent cerebral embolization, and/or triggering in situ thrombosis of cerebral vessels, plays a pathogenetic role in the neurologic manifestations of this disorder.

[Immunosuppressive therapy of myocarditis?]. / Immunsuppressive Therapie der Myokarditis?

Clinical and experimental data suggest that autoimmunological mechanisms may play an important role in the pathogenesis of postmyocardial cardiomyopathy. This may be due to the viral infection itself, or may be induced by persistence of the virus. On this basis immunosuppressive therapy aimed at preventing the progression of myocarditis to dilated cardiomyopathy is discussed. Apart from the clinical signs of myocarditis, which are usually not specific, the diagnosis of acute myocarditis should be enabled by the histological examination of endomyocardial biopsies. In accordance with the Dallas criteria the histological diagnosis of acute myocarditis is defined by the presence of inflammatory cells in the myocardium associated with myocyte necrosis and degeneration of adjacent myocytes. This morphology, however, is only seen within the first seven to ten days of the acute stage of the disease. Later, most cases of clinically suspected acute myocarditis are histopathologically consistent with the diagnosis of "borderline myocarditis" 1 h according to the Dallas classification. Although endomyocardial biopsies have markedly improved diagnostic possibilities, the diagnosis of myocarditis by light microscopy has its limitations. Thus, interobserver variability in the interpretation of biopsy samples remains high, and divergent frequencies of reported positive biopsies have continued to be published. Mainly the differentiation of infiltrating mononuclear cells from interstitial fibroblasts or pericytes is visually difficult. New immunohistochemical methods were therefore introduced to improve the specificity and sensitivity of the histological diagnosis. Using monoclonal antibodies against cell surface markers from lymphocytes (CD3, CD4, CD8) the identification, characterization and quantification of lymphocytic infiltrates in the myocardium was improved very significantly. Furthermore, the use of monoclonal antibodies against MHC-class-I and class-II-antigens provides further information about the immunological status of the myocardium. In conclusion, the use of these new immunohistological methods offers the possibility of establishing immunological criteria in addition to histological parameters permit a more accurate diagnosis and a better understanding of immune mechanisms possibly involved in the pathogenesis of myocarditis and dilated cardiomyopathy. Since the introduction of endomyocardial biopsies in the diagnosis of myocarditis several papers have reported trials of immunosuppressive therapy in histologically-proven myocarditis. However, none of these studies were randomised, and only a limited number of patients were treated. Neither the primary diagnosis of myocarditis nor the histology, evaluating the course of the disease was well defined, and therefore different from one study to another. Moreover, the regimens so far used for immunosuppressive therapy were not standardized, making it impossible to compare the various studies.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of four immunometric assays for myeloperoxidase using luminescent and colorimetric signal detection.

This communication presents four different assay systems for the determination of myeloperoxidase in body fluids. One is based on conventional chemiluminescence, two on luminescence-amplified enzyme measurement using either spiroadamantane-1,2-dioxetanes with alkaline phosphatase or luminol/peroxidase/4-iodophenol coupled with a peroxidase label. The assays covered the range 0-600 micrograms/l peroxidase. Established reference range for EDTA plasma from healthy volunteers gave an upper limit of 250 micrograms/l (95% confidence limits). Intra-assay coefficients of variation were less than 5% for all assays, as seen in compound precision profiles. Inter-assay variation was less than 10% throughout the whole concentration range. Kinetic curves for the light production were performed over a 2 hour period for the enhanced luminescence assays. Dynamic ranges of these assays were compared with the conventional colorimetric assay using 4-nitrophenyl phosphate--alkaline phosphatase. The assay was standardized using a commercially available myeloperoxidase preparation with defined enzymatic activity. The protein content was estimated and the laboratory standard compared and calibrated in mass units.

Long-term preservation of active luminous bacteria by lyophilization.

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCl) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4 degrees C. Luminous bacteria were resuscitated after six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.

Antibody-mediated enhancement of calcium permeability in cardiac myocytes.

Our study shows that antibodies, specific to the ADP/ATP carrier of the inner mitochondrial membrane, crossreact with the cell surface of cardiac myocytes, where the calcium channel seems to be the antigenic determinant. The antibodies enhanced the calcium current and suppressed its inactivation. Affinity-purified antibodies (IgG) exhibit an acute cytotoxic effect, which required extracellular calcium and was prevented by calcium channel blockers. Our findings suggest that antibody-mediated cytotoxicity results secondary to calcium overload caused by enhanced cellular calcium permeability, requiring no complement-dependent process.

An antibiotic-overproducing mutant of Streptomyces granaticolor with impaired differentiation.

A thy- tetracycline-resistant mutant of Streptomyces granaticolor was prepared by mutagenesis of the parental strain ETH 7437. The mutant exhibits a different morphology and an overproduction of granaticins. The ability to form an aerial mycelium and spores has been lost. The mutant cells have a round or an atypical shape and a thick cell wall, the membraneous system is enlarged by numerous mesosomes. Division septa are formed rarely. The mutant is more sensitive to both low and high temperature than the parental strain. The altered features are stably maintained for many generations. Ribosomal proteins of the mutant do not differ substantially from those of the original strain indicating that the mutant phenotype is not due to an alteration at the translational level.

Preparation and sequencing of the cloacin fragment of Streptomyces aureofaciens 16S RNA.

A fast method for isolation of a 3'-terminal fragment of Streptomyces aureofaciens 16S RNA was developed. The procedure involves reaction of 70S ribosomes with cloacin DF13 and subsequent fractionation of the reaction mixture by polyacrylamide gel electrophoresis. The cloacin fragment was eluted from the gel and used directly for 3'-end labeling with cytidine-3',5'-[5'-32P]bisphosphate. The labeled RNA fragment was sequenced by the enzymatic method. It consists of 50 nucleotides and has the sequence 5'-GUCGUAACAAGGUAACCGUACCGGA-AGGUGCGGUUGGAUCACCUCCUUUCOH. The differences from the E. coli and Bacillus sequences and their possible influence on the rate and specificity of polypeptide synthesis are discussed.

Gene rpmF for ribosomal protein L32 and gene rimJ for a ribosomal protein acetylating enzyme are located near pyrC (23.4 min) in Escherichia coli.

An Escherichia coli mutant harbouring altered ribosomal protein L32 has been isolated and genetically characterized. The mutation leading to this alteration (rpmF) and the temperature-sensitive mutation (ts-1517) present in the same strain were found to map near pyrC (23.4 min), being cotransducible not only with pyrC but also with fabD, flaT and purB in P1 phage mediated transductions. Furthermore, we found that the gene rimJ, which encodes an enzyme that acetylates the N-terminal alanine of protein S5 and the temperature-sensitive mutation, ts-386, present in the rimJ mutant strain (Cumberlidge and Isono 1979) also mapped in this region. Thus, the order of genes is deduced to be: ts-386-pyrC-ts-1517-rimJ-flaT-fabD-rpmF-purB+ ++.

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

Susceptibility of ribosomes of the tetracycline-producing strain of Streptomyces aureofaciens to tetracyclines.

Ribosomes from cells of Streptomyces aureofaciens producing tetracycline antibiotics (Tc-ribosomes) differ in electrophoretic mobility of ribosomal proteins S2, S10 and L19 from those of the same strain, where the production of tetracyclines was suppressed by changed cultivation conditions (C-ribosomes). Purified tight vacant couples C- and Tc-ribosomes are equally active in the translation of poly(U). Both types of S. aureofaciens ribosomes are more sensitive to tetracycline and chlortetracycline than ribosomes of Escherichia coli in the Phe-tRNA binding and the translation of poly(U).

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline.

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P0 cores lacking proteins L7/L12. Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state.

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.

Interaction of granaticin B with the transcription system of Bacillus subtilis.

The interaction of granaticin B, a quinone antibiotic produced by Streptomyces granaticolor, with some biologically important bivalent metal ions, DNA and ATP was demonstrated spectrophotometrically. The activity of isolated RNA polymerase was higher when the DNA of phage SP 50 served as template than with DNA isolated from Bacillus subtilis. Granaticin B inhibited in vitro RNA synthesis, similarly to certain other antibiotics (the inhibition was three times lower than that caused by actinomycin D or streptolydigin and slightly higher than that by epsilon-pyrromycinone). The inhibitory effect was higher when the Mg2+ concentration in the reaction mixture was decreased. The inhibition was then proportional to the concentration of the DNA template. DNA-dependent RNA synthesis is thus inhibited in vitro by granaticin B but this does not appear to be the only site of action of this antibiotic in vivo.