Application of an asymmetric helical tube reactor for fast identification of gene transcripts of pathogenic viruses by micro flow-through PCR.

We have established a fast PCR-based micro flow-through process consisting of a helical constructed tube reactor. By this approach we can detect transcripts of measles and human papilloma virus (HPV) by continuous flow allowing for reverse transcription (RT) and amplification of cDNA. The micro reaction system consisted of two columnar reactors for thermostating the different reaction zones of the RT process and the amplification. The PCR reactor was built by asymmetric heating sections thus realizing different residence times and optimal conditions for denaturation, annealing and elongation. The system concept is based on low electrical power consumption (50-120 W) and is suited for portable diagnostic applications. The samples were applied in form of micro fluidic segments with single volumes between 65 and 130 nL injected into an inert carrier liquid inside a Teflon FEP tube with an inner diameter of 0.5 mm. Optimal amplification for template lengths of 292 bp (lambda-DNA), 127 bp (measles virus) and 95 bp (HPV) was achieved by maximal cycle times of 75 s.

[Focal nodular hyperplasia of the liver]. / Fokalnoduläre Hyperplasie der Leber.

For several weeks a 27-year-old man had experienced a pressure sensation and noticed a spherical area of resistance in the epigastrium. On physical examination the firm, elastic tumour seemed most likely to be arising from the liver. Sonography recorded a 7.4 x 10.5 cm echo-poor structure. Computed tomography demonstrated in the tumour a central star-like, hypodense zone which filled with contrast medium, a finding typical of focal nodular hyperplasia. Functional hepatobiliary scintigraphy and histological examination of a fine-needle biopsy from the tumour (slightly enlarged hepatocytes, portal reticular fibrosis, ductular proliferation) confirmed the diagnosis. As there was no danger of rupture and no sign of local compression surgical intervention was not indicated, but regular ultrasound monitoring is planned.

Effects of hypercholesterolemia on the nucleotide content in human blood cells.

In hypercholesterolemia significant changes in the nucleotide pattern of erythrocytes and lymphocytes as determined by high performance liquid chromatography were found. The decrease in ATP of lymphocytes in hypercholesterolemia from 10.4 +/- 0.3 to 7.0 +/- 0.4 nmol mg-1 protein (n = 8) was associated with an increase in ADP from 2.2 +/- 0.2 to 4.0 +/- 0.2 nmol mg-1 protein (P less than 0.005). The pattern of guanosine phosphates likewise was found to be changed in hypercholesterolemia. Akin to lymphocytes, red blood cells displayed marked changes in nucleotide levels. No such changes were observed in platelets. Cultured lymphocytes incubated with human plasma low density lipoproteins (LDL) (140 mg cholesterol dl-1) displayed a reversible fall in ATP and an increase in ADP by about 40% and 160%, respectively, with high density lipoproteins (HDL) or very low density lipoproteins (VLDL) being essentially ineffectual. It is concluded that in hypercholesterolemia a significant change in the nucleotide pattern of blood cells is exerted by the increase in LDL. Possible pathophysiological implications are discussed.

Hormonal regulation of calcium current in freshly isolated airway smooth muscle cells.

Single freshly isolated smooth muscle cells of adult bovine trachea were voltage clamped, and the calcium inward current was separated from K+ currents by blocking the large outward currents with intra- and extracellular Cs+ and extracellular tetraethylammonium chloride. Isoproterenol stimulated peak calcium current (ICa) in a dose-dependent manner through the beta-adrenergic receptor. The isoproterenol effect was not mediated or caused by the stimulation of a K+ or Na+ current, a decrease in the intracellular concentrations of Ca2+ or H+, the stimulation of the Na(+)-H+ or the Na(+)-Ca2+ exchanger. Neither basal nor isoproterenol-stimulated ICa was affected by internal dialysis of the cell with adenosine 3',5'-cyclic monophosphate (cAMP), cAMP analogues, or the catalytic subunit of cAMP-kinase. Internal dialysis of the cells with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the stimulation of isoproterenol whereas dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) induced an isoproterenol-like maximal increase of ICa. These results show that the beta-adrenergic receptor stimulates the L-type calcium current of isolated tracheal smooth muscle cells independent of cAMP and cAMP-kinase through a GTP/GDP regulated protein.

Familial synovial chondromatosis combined with dwarfism.

In a family with synovial chondromatosis affecting at least three, presumably five members, the articular disorder was combined with dwarfism. The persons with joint disorders were below the third percentile in body height, but family members with normal articular function had normal height. We believe this to be the first description of a combination of synovial chondromatosis with genetically caused dwarfism.

Effects of human low-density lipoproteins on the nucleotide patterns of cultured pig aortic endothelial cells.

Cultured pig aortic endothelial cells display significant changes in their nucleotide patterns after incubation with LDL-cholesterol purified from normal human plasma as determined by HPLC. Incubation at 70 mg/dl LDL-cholesterol for 24 h at 37 degrees C caused a significant decrease (P less than 0.001) in ATP from a control value of 14.0 +/- 0.4 nmol/mg protein to 6.6 +/- 0.9 nmol/mg protein (n = 4) with a concomitant increase in ADP and AMP. At higher LDL concentrations these effects were even more pronounced but still reversible. Akin to adenine nucleotides, the guanosine and uridine phosphates as determined by HPLC were changed. In contrast to LDL, HDL and VLDL were ineffectual.

Muscle metabolism and red cell ATP/ADP concentration during bicycle ergometer in patients with AMPD-deficiency.

6 patients with AMPD-deficiency and 6 control subjects performed exercise on a bicycle ergometer until heart rate was 200 minus age. During exercise the increase of ammonia plasma concentrations was reduced in AMPD-deficient patients compared with that of control subjects. Plasma concentrations of lactate, pyruvate, inosine, hypoxanthine and xanthine increased during exercise in both groups. The concentrations of lactate, inosine and hypoxanthine were lower in AMPD-deficient patients during exercise, the difference was not significant. In AMPD-deficient patients the ATP-concentrations of red blood cells increased during exercise in contrast to control subjects, whereas the ADP amount did not change significantly. Our data suggest that in AMPD-deficient-patients AMP is mainly reduced to adenosine during exercise resulting in decreased ammonia concentrations. The increased concentrations of ATP in red blood cells may be the consequence of increased phosphorylation.

Beta-adrenergic receptor stimulates L-type calcium current in adult smooth muscle cells.

The hormonal regulation of L-type calcium current was investigated in freshly isolated tracheal smooth muscle cells using the whole-cell configuration of the patch-clamp technique. Isoproterenol stimulated the L-type calcium current 2.6-fold through beta-adrenoceptors. Dialysis of these cells with cyclic AMP, cyclic AMP analogues or the catalytic subunit of cyclic AMP kinase had no effect on basal or isoproterenol-stimulated calcium current. The calcium current was stimulated and inhibited by dialysis of the cells with GTP gamma S and GDP beta S, respectively. Evidently, in some smooth muscle cells the beta-adrenoceptor couples directly to L-type calcium channels via a G protein.

Non-excretory plasmocytoma--value of imaging procedures.

We report a case of a 84-year old patient with a non-excretory plasmocytoma. Lacking pathological laboratory values, first diagnostic hints on this rare disease were provided by imaging procedures. Computed tomography and plain radiography surpassed bone scan with 99mTc-diphosphonate compounds in detecting bone associated lesions. According to data in the literature, magnetic resonance imaging and bone marrow scintigraphy seem to be useful in quantifying bone marrow involvement, whereas computed tomography and plain radiography appear to be good tools in detecting localized cortical or spongious lesions in multiple myeloma.

Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea.

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.