Age-related nonrandom chromosomal abnormalities in human low-grade astrocytomas

We report a cytogenetic investigation of 55 low-grade astrocytomas in 52 patients, 15 children and 37 adults. In addition to numerical aberrations such as trisomy 7 and gonosomal losses, we found structural and/or numerical aberrations of chromosome 1 in eight astrocytomas. There was a striking difference between the rearranged chromosomes in pediatric and adult patients. Whereas the pediatric tumors revealed monosomies 1p with accompanying trisomy 1q, the astrocytomas in adults showed partial or complete monosomies 1q.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Mutagenicity of 5,6-dimethoxysterigmatocystin, a metabolite from Aspergillus multicolor, in the Salmonella/microsome system.

The natural sterigmatocystin derivative, 5,6-dimethoxysterigmatocystin, was found to be a mutagen for Salmonella typhimurium strains TA98 and TA100 after metabolic activation in a mammalian microsome system.     

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding

The nerve growth factor (NGF) receptor is a glycosylated transmembrane protein present on the cell surface as both high and low affinity forms, but biological responsiveness requires interactions of NGF with the high affinity site. We have tested the effects of mutations in the intracellular domain of the receptor upon its cell surface expression and equilibrium binding of 125I-NGF. Although mutant receptors lacking the entire cytoplasmic domain are processed and expressed at the cell surface and are capable of binding to NGF, the absence of cytoplasmic sequences leads to a loss of high affinity binding and to a lack of an appropriate cross-linking pattern as assessed by N-hydroxysuccinimidyl 4-azidobenzoate photoaffinity cross-linking. These results, taken together with the highly conserved nature of these cytoplasmic sequences, implies that the interaction of the receptor with an accessory molecule is necessary to form the high affinity receptor.     

Quantitative 125J-Autoradiographie einzelner Zellen

Zusammenfassung Das nach der Energie seiner -Zerfälle zwischen Tritium und Kohlenstoff-14 liegende Isotop ¹²⁵J wurde auf seine Eignung zur quantitativen Autoradiographie geprüft. Absorption und Geometrie-Faktoren der radioaktiven Strahlung wurden untersucht. Hieraus ließen sich geeignete Meßbedingungen entwickeln. Durch gleichzeitige Exposition von radioaktiven Referenzquellen können absolute Radioaktivitätsmengen ermittelt werden. Als Referenzquellen sind membranmarkierte Standardzellen geeignet, die den physikalischen Eigenschaften des Isotopes Rechnung tragen. Hierzu wurden enzymatisch radiojodinierte Schaferythrozyten auf ihre Eignung geprüft. Die absolute Zahl von antigenen Stoffen auf den Oberflächen einzelner Zellen erhält man, wenn man die spezifische Aktivität der markierten Antikörpermoleküle bestimmt und die Silberkorndichte der zu untersuchenden Zellen und der Standardzellen mißt. Die Radioaktivität pro Standardzelle wird in herkömmlicher Weise ermittelt.Die neue Methode wurde zur Quantifizierung membrangebundener Immunglobulinmoleküle vom IgG-Typ auf einzelnen menschlichen Lymphozyten angewendet. Hierbei ist die Ermittlung einer immunologischen Sättigung des markierten Antikörpers wesentlich. Auf Lymphozyten von Normalpersonen und von chronisch-lymphatischen Leukämiepatienten konnten sehr unterschiedliche absolute Immunglobulinmengen bestimmt werden.

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Quantitative ¹²⁵I-autoradiography of individual cells

Summary Iodine 125, an emitter of -radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures.The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.

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