[Differential Diagnosis of Mediastinal and Hilar Lymphadenopathy with Focus on Occupational Diseases]. / Differenzialdiagnose bei mediastinaler und hilärer Lymphadenopathie mit Fokus auf berufsbedingte Krankheitsbilder.

Hilar and mediastinal lymphadenopathy may represent a diagnostic challenge in clinical practice. This article is intended to facilitate differential diagnosis by a systematic description of relevant pathologies, notably with occupational etiology. Clinical findings of relevant diseases, i. e. of tuberculosis, chronic beryllium disease, sarcoidosis, lung cancer, malignant lymphoma, Epstein-Barr virus infection, and histoplasmosis are compared.Case history, imaging and laboratory tests have important diagnostic impact. But also invasive methods can be necessary in order to exclude and prove malignancy, infection or autoimmune disease.

Soil warming alters nitrogen cycling in a New England forest: implications for ecosystem function and structure.

Global climate change is expected to affect terrestrial ecosystems in a variety of ways. Some of the more well-studied effects include the biogeochemical feedbacks to the climate system that can either increase or decrease the atmospheric load of greenhouse gases such as carbon dioxide and nitrous oxide. Less well-studied are the effects of climate change on the linkages between soil and plant processes. Here, we report the effects of soil warming on these linkages observed in a large field manipulation of a deciduous forest in southern New England, USA, where soil was continuously warmed 5°C above ambient for 7 years. Over this period, we have observed significant changes to the nitrogen cycle that have the potential to affect tree species composition in the long term. Since the start of the experiment, we have documented a 45% average annual increase in net nitrogen mineralization and a three-fold increase in nitrification such that in years 5 through 7, 25% of the nitrogen mineralized is then nitrified. The warming-induced increase of available nitrogen resulted in increases in the foliar nitrogen content and the relative growth rate of trees in the warmed area. Acer rubrum (red maple) trees have responded the most after 7 years of warming, with the greatest increases in both foliar nitrogen content and relative growth rates. Our study suggests that considering species-specific responses to increases in nitrogen availability and changes in nitrogen form is important in predicting future forest composition and feedbacks to the climate system.

Efficacy of enzymatic toothpastes for immobilisation of protective enzymes in the in situ pellicle.

AIM: Different enzyme-containing toothpastes are available on the market. The aim of the present in situ study was to investigate their efficacy for immobilisation of protective enzymes in the pellicle layer. METHODS: Pellicle formation took place in situ on bovine enamel slabs fixed to individual upper jaw splints carried by 6 subjects. After pellicle formation for 1 min, brushing was performed for 3 min with the commercially available toothpastes Enzycal, biotène and BioXtra, respectively. Before as well as 0, 20 and 40 min after brushing, samples were removed from the splints and tested for lysozyme, peroxidase and glucoseoxidase activity. The assays for the respective enzyme activities were based on fluorogenic substrates. Separate experiments were conducted for the different enzymes and toothpastes. RESULTS: Brushing with the toothpastes caused an extensive increase of glucoseoxidase activity in the pellicle, but it was of low tenacity whereas peroxidase activity was enhanced considerably. However, targeted accumulation of lysozyme in the pellicle was not very pronounced. Brushing without toothpaste had no effect on enzyme activities in the acquired pellicle. CONCLUSION: Targeted immobilisation of enzymes in the in situ pellicle can be achieved with toothpastes.

Soil warming and carbon-cycle feedbacks to the climate system.

In a decade-long soil warming experiment in a mid-latitude hardwood forest, we documented changes in soil carbon and nitrogen cycling in order to investigate the consequences of these changes for the climate system. Here we show that whereas soil warming accelerates soil organic matter decay and carbon dioxide fluxes to the atmosphere, this response is small and short-lived for a mid-latitude forest, because of the limited size of the labile soil carbon pool. We also show that warming increases the availability of mineral nitrogen to plants. Because plant growth in many mid-latitude forests is nitrogen-limited, warming has the potential to indirectly stimulate enough carbon storage in plants to at least compensate for the carbon losses from soils. Our results challenge assumptions made in some climate models that lead to projections of large long-term releases of soil carbon in response to warming of forest ecosystems.

Ether--lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether--lipid analogues in Leishmania.

Ether-lipid (alkyl-phospholipid) analogues such as Miltefosine possess potent in vitro and in vivo anti-leishmanial activity and these compounds are currently undergoing clinical trials in humans. These analogues are also effective against Trypanosoma cruzi and Trypanosoma brucei subspecies but their mode of action is not known. Leishmania have high levels of ether-lipids and these are mainly found in the glycosylphosphatidylinositol-anchored glycolipids and glycoproteins present on the surface of the parasites. In Leishmania mexicana promastigotes we have studied both the initiating steps for the biosynthesis of ether-lipids, and key remodelling steps. The effect of Miltefosine and Edelfosine, on key enzymes involved in the metabolism of ether-lipids has been studied. The enzymes include dihydroxyacetonephosphate acyltransferase, sn-l-acyl-2-lyso-glycero-3-phosphocholine and sn-l-alkyl-2-lyso-glycero-3-phosphocholine acyltransferases. We confirm that the initiating steps in ether-lipid metabolism in Leishmania are present in glycosomes, and that Miltefosine or Edelfosine did not perturb these enzymes. The metabolism of the latter phosphatidylcholine base intermediates, which may be involved in the remodelling of acyl- and alkyl-glycerophospholipids, was also seemingly associated with glycosomes. Both Miltefosine and Edelfosine inhibited this microbody (glycosomal) located alkyl-specific-acyl-CoA acyltransferase in a dose-dependent manner with an inhibitory concentration of 50 microM. It is suggested therefore that a perturbation of ether-lipid remodelling could be responsible for the anti-leishmanial action of these drugs.

Ca2+ and Na+ permeability of high-threshold Ca2+ channels and their voltage-dependent block by Mg2+ ions in chick sensory neurones.

1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] (< 10(-8) M) and with Na+o and Cs+i as the main charge carriers (120 mM), HVA Na+ currents started to activate at -40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3. Addition of 30-500 microM Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD = 39 microM in 120 mM Na+ at -10 mV). The block was maximal at negative potentials (< -70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4. Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD = 24 mM in 2 mM Ca2+ at -30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I-V relationships on return from very positive potentials (+100 mV). 5. Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg2+ was too fast (< 100 microseconds) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990).

Characterisation of phosphoglycerate kinase genes in Leishmania major and evidence for the absence of a third closely related gene or isoenzyme.

We have characterised the phosphoglycerate kinases (PGKs) in L. major and studied their mRNA and protein expression. Interestingly we have found evidence for only two tandemly linked PGK genes which correspond to the PGK gene B and C homologue in Trypanosoma and Crithidia. The primary structure of the leishmanial PGK genes B and C are virtually identical and differed only by the presence of a 62 amino acid extension at the carboxyl terminal of the PGK gene C homologue which is therefore likely to contain the translocation signal for glycosomal topogenesis. Indeed, the PGK gene C protein was found to be glycosomal (gPGK) while the PGK gene B protein was found to be cytosolic (cPGK). Both PGK genes are expressed in L. major promastigotes with the cPGK transcript expressed at a much higher level (4-5-fold) than the gPGK transcript. Similarly the relative cPGK isoenzyme activity was found to be approximately 4-fold higher than that of the gPGK isoenzyme. Surprisingly in L. major we have found no evidence for the PGK gene A present in all other trypanosomatids studied to date (Trypanosoma brucei, Trypanosoma congolense and Crithidia fasciculata). We therefore consider the possible evolutionary and functional significance of a trypanosomatid with only two PGK isoenzymes.

Fast local superfusion technique.

A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 micron diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the deadspace of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions. By increasing the pressure in the superfusion pipette, the laminar stream between the pipettes was forced down onto the cell layer. The change from bath to superfusion solutions, evaluated by liquid junction potential changes, occurs in the order of 1 ms.

Different effects of baclofen and GTP gamma S on voltage-activated Ca2+ currents in rat hippocampal neurons in vitro.

Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.

Growth cone calcium ion channels: properties, clustering, and functional roles.

Voltage-dependent Ca2+ channels appear to constitute a central component of signal transduction cascades in neuronal growth cones. By means of spatially selective superfusion in the subcellular range growth cone, Ca2+ channels were investigated quantitatively. Both principal types of Ca2+ channels, low voltage activated and high voltage activated Ca2+ channels were present in growth cones of cells regenerating neurites in culture as well as growth cones of differentiating neuronal precursor cells. Studies concerning their spatial distribution revealed a remarkable clustering of Ca2+ channels at the growth cone. Their possible functional roles in neurite growth, axonal pathfinding, and synaptogenesis are discussed.

Glutamate-produced long-term potentiation by selective challenge of presynaptic neurons in rat hippocampal cultures.

Excitatory postsynaptic long-term potentiation (LTP) was observed after restricted glutamate or metabotropic agonist application to somata and proximal neurites of identified presynaptic neurons. LTP-induction in this way entailed delays of minutes that correlated with lengths of afferent fibres. Potentiation failed to occur in cells pretreated with colchicine to degrade their microtubular transport matrix. The results suggest fast axonal transport to be a message carrier in the acquisition process of LTP.

Augmentation of calcium channel currents in response to G protein activation by GTP gamma S in chick sensory neurons.

G protein-mediated downregulation of current through neuronal voltage-gated Ca2+ channels is well known. We now report that G protein activation by GTP gamma S increases the Ba2+ conductance of high-voltage-activated Ca2+ channels of chick dorsal root ganglion (DRG) cells. This occurs with a delay of minutes during which the channels are inhibited by the activated G proteins. The Ba2+ current (IBa) showed an absolute enhancement by a factor near 2, 15 min after GTP gamma S application. However, by utilizing prior observations of the voltage dependence of the inhibitory action we could demonstrate that the G protein-inhibited component of IBa, was still present. Moreover, the achieved amount of IBa disinhibition showed little variation throughout the experiments. This indicates that the increase in IBa is not due to a relief of the inhibitory action of activated G proteins but to the slow appearance of a distinct upregulating action, probably through a different pathway. Augmentation of IBa was eliminated by pertussis toxin (PTX) infusion or pretreatment, but was also prevented by intracellularly infusing protein kinase C (PKC) inhibitors together with GTP gamma S. The upregulation of neuronal Ca2+ channels thus appears to be exerted through a messenger pathway upstream of PKC activation that involves G proteins. Augmentation of Ca2+ currents (ICa) was observed only with strong intracellular [Ca2+] buffering, which suggests a control of the upregulating action by even moderate increase in intracellular [Ca2+].

Constitutive upregulation of calcium channel currents in rat phaeochromocytoma cells: role of c-fos and c-jun.

1. Northern blot analysis and cell transfection were used in conjunction with whole-cell current recordings to examine the involvement of the immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were exposed to nerve growth factor (NGF) and to depolarizing concentrations of KCl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically retained their undifferentiated morphology even after 5 days of treatment. 3. The maximal amplitude of high-voltage-activated calcium currents (ICa) increased from the control level of 117.8 +/- 48.3 (mean +/- S.D.) to 387.2 +/- 90.1 pA within 3 days of NGF treatment. omega-Conotoxin (5-10 microM) inhibited 24.6 +/- 8.5% of ICa in undifferentiated cells and 57.8 +/- 6.9% in NGF-treated cells. 4. The levels of c-fos and c-jun mRNAs increased transiently during each daily exposure to NGF. The level of c-fos mRNA also increased transiently during repeated KCl-induced depolarizations but c-jun mRNA remained low or absent. 5. Naive PC12 cells were transiently co-transfected with expression plasmids that contained the full length of c-fos and c-jun cDNA. After 2 days following transfection, the PC12 cells could be grouped according to the size of ICa. In 56% of cells, ICa was similar to control currents (106.1 +/- 37.4 pA). In the remaining 44% of cells, ICa showed a 2.2-fold enhancement with respect to control cells. Transfection of only c-fos had no effect on ICa but, in 24% of cells transfected with c-jun, ICa was 176.6 +/- 124.6 pA. Since periodic membrane depolarization induced c-fos but not c-jun mRNA, c-jun transfection was combined with a high-K+ treatment over 3 days. In 18% of treated cells, ICa was 3.7 times larger than control currents. Morphological differentiation was not observed in transfected cells. 6. In PC12 cells co-transfected with c-fos and c-jun or treated with high K+ after transfection of c-jun, omega-conotoxin (5-10 microM) inhibited 68.7 +/- 11.9% of ICa when the current amplitude was in the range of 200-600 pA. since similar concentrations of omega-conotoxin blocked 19.2 +/- 5.4% of ICa in control cells, the current increase induced by c-fos and c-jun was supported by up to 11-fold enhancement of the omega-conotoxin-sensitive component of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

The formation of glutamatergic synapses in cultured central neurons: selective increase in miniature synaptic currents.

The formation of synapses between cultured rat thalamic neurons was studied with electrophysiological and immunocytochemical methods. Thalamic neurons in culture form predominantly glutamatergic synapses. Already after 3 days in vitro glutamatergic miniature EPSCs occurred spontaneously and their frequency was strongly increased after K+ depolarization, while GABAergic mIPSCs were found after K+ depolarization at lower frequency. This demonstrates that both, excitatory glutamatergic and inhibitory GABAergic synapses were functional in close succession to initial neurite outgrowth. Synapses formed independent of spontaneous electrical activity, which was absent during the first week in culture. Spontaneous action potentials appeared during the second week and chronic action potential blockade by addition of tetrodotoxin reduced neuronal survival and the number of glutamatergic synapses per neuron. During in vitro differentiation the number of synapsin I immunoreactive presynaptic terminals and the frequency of spontaneous glutamatergic miniature EPSCs increased closely correlated, while the frequency of GABAergic mIPSCs after K+ depolarization did not increase. Thus, the continous formation of presynaptic terminals, including possible maturation of transmitter release, appeared to underlie the increase in mEPSC frequency. Analysis of miniature EPSC amplitudes at different stages in vitro revealed an increase in amplitudes, suggesting synaptic differentiation after initial establishment of functional transmission in glutamatergic synapses. This process was synapse specific as amplitudes of GABAergic mIPSCs were invariant.

Enhancement of voltage-gated Ca2+ currents induced by daily stimulation of hippocampal neurons with glutamate.

The regulation of calcium channel currents (ICa) induced by daily stimulation (1 hr) with 10 microM glutamate was studied in full differentiated hippocampal cells in culture. We report a specific enhancement of the high-voltage-activated current type (HVA ICa) ongoing over days. The density of HVA ICa increased about twofold after the second glutamate session, and this enhancement was still observed after the fifth day of treatment, while low-voltage-activated calcium currents (LVA ICa) remained unchanged. During glutamate application, a transient increase of intracellular calcium (Cai) was observed, followed by a slow decay within 2-3 min, and substantial recovery in about 10 min. Similarly, Cai transients induced by periodic membrane depolarization mimicked the long-term effect of glutamate on ICa. These results demonstrate for the first time an increase of ICa in a time frame of days. Since the effect of glutamate on ICa was prevented by cycloheximide, neosynthesis of channel proteins presumably supports this enhancement.

Kinetics of GABAB receptor-mediated inhibition of calcium currents and excitatory synaptic transmission in hippocampal neurons in vitro.

The time courses of the gamma-aminobutyric acid type B (GABAB) receptor-mediated inhibition of excitatory synaptic transmission and of action potential-evoked calcium currents were studied in hippocampal neurons in vitro with step-like changes of a saturating baclofen concentration. Inhibition mediated by postsynaptic GABAB receptors was excluded pharmacologically. Both presynaptic inhibition and reduction of calcium currents developed and declined exponentially with similar time constants of about 0.2 and 3 s, respectively. The close correlation of the time courses indicates that fast, G protein-mediated depression of voltage-gated calcium channels and thus direct reduction of the presynaptic calcium influx may contribute to the GABAB receptor-induced inhibition of excitatory synaptic transmission in hippocampal neurons in vitro.

NH4Cl-induced inward currents and cytoplasmic Ca2+ transients in chick sensory neurones.

In neurones cultured from chick dorsal root ganglia, application of NH4Cl (3-45 mM) produced a transient inward current followed by a sustained current at negative holding potentials. Methylamines, hydrazine and guanidine were not able to mimic the effects of NH4Cl. The transient, but not the steady current, was inactivated during successive applications of NH4Cl. Challenge with acidic solutions (pH 6.0-6.4) or Ca(2+)-free solutions induced similar currents and abolished NH4Cl-induced transients. Exposure to 10 mM NH4Cl transiently increased cytoplasmic free Ca2+, which then fell to a sustained plateau. NH4Cl-induced membrane depolarization and the concomitant elevation in intracellular Ca2+ can play an important role in modulation of neuronal activity.

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons.

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca2+ concentration, [Ca]i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]i. During wash-out [Ca]i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]i was quickly restored. [Ca]i recovery was delayed after treating the cell with 2 microM thapsigargin, an inhibitor of the Ca2+ pump of internal Ca2+ stores. Caffeine (10 mM) transiently increased [Ca]i. A second caffeine application produced smaller [Ca]i changes due to the prior depletion of Ca2+ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 microM) transiently increased [Ca]i both in the standard and Ca(2+)-free solution. [Ca]i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La3+ and in Ca(2+)-free solutions, but which was greatly diminished in Na(+)-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]i transients and in the inward current induction.

Whole cell and single channel analysis of the kinetics of glycine-sensitive N-methyl-D-aspartate receptor desensitization.

1. The kinetics of glycine-sensitive, N-methyl-D-aspartate (NMDA) receptor desensitization were investigated in cultured neurones with the patch clamp technique. 2. The degree of fast NMDA-receptor desensitization was inversely related to glycine concentration. Thus, increasing concentrations of glycine from 30 nM to 2.5 microM potentiated desensitized NMDA responses (873% +/- 101%) to a greater degree than peak responses (260% +/- 27%). 3. The desensitization was due to a decrease in the affinity of glycine for the strychnine-insensitive, glycine modulatory site (glycineB site) following activation of the NMDA-receptor complex. Thus, the A50 for glycine in potentiating peak responses (77 nM, 95% confidence limited 58-104 nM) was five fold lower than that for plateau responses (399 nM, 340-468 nM). 4. The rate of desensitization was related to glycine concentration such that a reciprocal plot of desensitization rate (1/tau S-1) against glycine concentration had a slope of 9.5* 10(6) M-1 S-1. 5. Recovery from desensitization following step increases in glycine or L-alanine concentration in the continuous presence of NMDA (200 microM) reflected the association kinetics of the glycineB agonist used. 6. The rate and degree of NMDA receptor desensitization was independent of holding potential. 7. NMDA receptor desensitization was also evident at the single channel level. 8. The glycineB antagonist 7-chlorokynurenic acid (7-Chl-Kyn 3 and 10 microM) concentration-dependently induced an identical form of desensitization in the presence of 1 microM glycine. 9. In contrast, the competitive NMDA antagonist (+/-)-amino-phosphonovaleric acid (APV 30 to 300 microM) concentration-dependently antagonized and slowed the onset kinetics of NMDA responses.