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Biomedical subjects

J Behr

Publications and source records attributed to J Behr.

At least 73 records · Page 4Linked to original sources
PubMed1998

Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling.

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates. A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.

5-Aminolevulinate Synthetase↗
PubMed1998

Gene transfer with lipospermines and polyethylenimines.

It is an obvious and basic principle that to be efficient, gene therapy requires effective gene transfer followed by adequate gene expression. However, getting DNA, a pro-drug, into the cell and into the nucleus, remains a crucially limiting factor. Even recombinant viral methods still show poor performances in clinical situations and non-viral methods are considered classically to be of yet lower efficiency. Here, we consider the mode of action, the nature of the complexes formed with DNA and the transfection potentials of two categories of inert, cationic vectors, the lipospermines and polyethylenimine. Both are among the best vectors currently available for in vitro work. Moreover, polyethylenimine is proving to be a versatile and effective carrier for different in vivo situations, especially for delivering genes into the mammalian brain.

Journal Article↗
PubMed1998

Subthreshold membrane potential oscillations in neurons of deep layers of the entorhinal cortex.

Neuronal oscillations are important for information processing. The entorhinal cortex is one of the structures which is involved in generation of theta rhythm. The major role of the entorhinal cortex is to feed diverse sources of information both to and from the hippocampus. Far from simply being a funnel for this information it becomes clear that the entorhinal cortex has its own active properties that contribute to signal processing. Interestingly, stellate cells in layer II of the entorhinal cortex can intrinsically generate subthreshold, Na+-dependent membrane potential oscillations. Here, using intracellular and patch-clamp recordings, we report a similar phenomenon from neurons of the deep layers of the entorhinal cortex. In our in vitro slice preparation about two-thirds of recorded neurons were able to generate voltage-sensitive subthreshold membrane potential oscillations. At a membrane potential of about 50 mV the mean frequency of the voltage-oscillations was 8.1 Hz, whereby at slightly more positive potentials (-44 mV) the frequency of the membrane potential oscillations was 20 Hz and the oscillations became interrupted by clusters of non-adapting trains of spikes. Pharmacological experiments revealed that the oscillations were not affected by Cs+, but could be blocked by the fast Na+-channel blocker tetrodotoxin. We therefore conclude that voltage- and Na+-dependent subthreshold membrane potential oscillations are not only present in stellate cells of entorhinal cortex-layer II, but are also typical for neurons of the deep layers of the entorhinal cortex.

Animals↗
PubMed1998

The perforant path projection from the medial entorhinal cortex layer III to the subiculum in the rat combined hippocampal-entorhinal cortex slice.

Intracellular recordings were performed to examine the perforant path projection from layer III of the entorhinal cortex to the subiculum in rat combined hippocampal-entorhinal cortex slices. Electrical stimulation in the medial entorhinal cortex layer III caused short latency combined excitatory and inhibitory synaptic responses in subicular cells. In the presence of the GABA(A) antagonist bicuculline and the GABA(B) antagonist CGP-55845 A inhibition was blocked and isolated AMPA- or NMDA receptor-mediated EPSPs could be elicited. After application of the non-NMDA antagonist NBQX and the NMDA antagonist APV excitatory responses were completely blocked indicating a glutamatergic input from the neurons of the medial entorhinal cortex layer III. By stimulation from a close (< 0.2 mm) position in the presence of NBQX and APV and either CGP-55845 A or bicuculline we could record monosynaptic fast GABA(A) or slow GABA(B) receptor-mediated IPSPs, respectively. We compared synaptic responses in subicular cells induced by stimulation in the medial entorhinal cortex layer III with responses elicited by stimulation of afferent fibres in the alveus. The EPSPs of subicular cells induced by stimulation of alvear fibres could be significantly augmented by simultaneous activation of perforant path fibres originating in the medial entorhinal cortex layer III, while delayed activation of alvear fibres after stimulation of the perforant path resulted in a weak inhibition of the alveus evoked EPSPs. Thus, the perforant path projection activates monosynaptic excitation of subicular neurons. Therefore the entorhinal cortex does not only function as an important input structure of the hippocampal formation but is also able to modulate the hippocampal output via the entorhinal-subicular circuit.

Animals↗
PubMed1998

Cytochrome c oxidase: structure and spectroscopy.

Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water. Electron transfer is coupled to proton translocation across the membrane, resulting in a proton and charge gradient that is then employed by the F0F1-ATPase to synthesize ATP. Over the last years, substantial progress has been made in our understanding of the structure and function of this enzyme. Spectroscopic techniques such as EPR, absorbance and resonance Raman spectroscopy, in combination with site-directed mutagenesis work, have been successfully applied to elucidate the nature of the cofactors and their ligands, to identify key residues involved in proton transfer, and to gain insight into the catalytic cycle and the structures of its intermediates. Recently, the crystal structures of a bacterial and a mitochondrial cytochrome c oxidase have been determined. In this review, we provide an overview of the crystal structures, summarize recent spectroscopic work, and combine structural and spectroscopic data in discussing mechanistic aspects of the enzyme. For the latter, we focus on the structure of the oxygen intermediates, proton-transfer pathways, and the much-debated issue of how electron transfer in the enzyme might be coupled to proton translocation.

Animals↗
PubMed1998

Enhanced propagation of epileptiform activity through the kindled dentate gyrus.

Extracellular recordings were performed in combined hippocampal-entorhinal cortex (HC-EC) slices obtained from control and commissural kindled rats to investigate the propagation of epileptiform activity from the entorhinal cortex (EC) to the hippocampus (HC) after chronic epilepsy. Lowering extracellular Mg2+ concentration in control slices induced epileptiform activity consisting of spontaneous epileptiform bursts in area CA3 and of electrographic seizures in the EC. In contrast, the CA3 region of HC-EC slices obtained from kindled rats displayed significantly longer lasting epileptiform bursts and electrographic seizures. The electrographic seizures that were absent in controls propagated from the EC because disconnecting the HC from the EC stopped their occurrence in the CA3, whereas epileptiform bursts persisted with an unaltered pattern and frequency. Thus the area CA3 is affected by kindling and contributes to the spread of epileptiform activity within the EC-HC complex. We developed a method to induce focal epileptiform activity in the EC by locally perfusing the gamma-aminobutyric acid-A (GABA) antagonist bicuculline (50 mM) in 10 mM KCl containing artificial cerebrospinal fluid. This method enabled us to investigate the propagation of epileptiform discharges from the disinhibited EC to the DG without affecting the DG with the epileptogenic medium. We show here that kindling facilitates the propagation of epileptiform activity through the DG. These data are consistent with the normal function of the DG as a filter limiting the spread of epileptiform activity within the HC-EC complex. This gating mechanism breaks down after chronic epilepsy induced by kindling.

Animals↗
PubMed1998

Preliminary studies of a novel oral fluoropyrimidine carbamate: capecitabine.

PURPOSE: To evaluate the toxicology and pharmacology of an orally active fluoropyrimidine given as a continuous daily dose divided into two portions for 6 weeks, and to determine the maximal-tolerated daily dose (MTD) and the suggested phase II daily dose. PATIENTS AND METHODS: Solid-tumor patients with a Karnofsky performance status greater than 70 who had normal organ function and resolution of the effects of prior therapy, and who gave informed written consent, were enrolled. Oral capecitabine, as a divided morning and evening dose, was administered to cohorts of a minimum of 3 patients starting at 110 mg/m2 and escalating by means of a modified Fibonacci scheme to 1,657 mg/m2/d. Pharmacologic samples were obtained on days 1 and 15. Toxicity evaluations were performed approximately every 3 days for the first 43 days. Antitumor effect was evaluated at day 42 of therapy. RESULTS: Thirty-three patients entered the study. Few side effects occurred at or below 1,331 mg/m2/d. The MTD was 1,657 mg/m2/d with limiting toxicities of palmar-plantar erythrodysesthesia, nausea, vomiting, vertigo, abdominal pain, diarrhea, and thrombocytopenia. All toxicities were reversible. A mixed response was seen in one breast cancer patient. Pharmacologic studies showed rapid and extensive metabolism of the parent drug into cytotoxic metabolites with a maximum plasma concentration (Cmax) 1 hour after ingestion. Linear increases in the area under the concentration-time curve (AUC) and Cmax were seen with linear increases in administered dose. CONCLUSION: The suggested phase II dose on a continuous 42-day dosing schedule is 1,331 mg/m2/d. Linear pharmacologic parameters of the parent compound and metabolites are demonstrated.

Administration, Oral↗
PubMed1997

Effects of serotonin on synaptic and intrinsic properties of rat subicular neurons in vitro.

Intracellular recordings were performed to study the effects of 5-HT on membrane properties and EPSP/IPSP responses of subicular neurons in rat combined hippocampal-entorhinal cortex slices. Application of 5-HT induced in 76% of the investigated subicular cells a hyperpolarization and a reduction of membrane resistance. In bursting neurons, 5-HT caused a reduction of the depolarizing envelope underlying burst discharges and attenuated the subsequent afterhyperpolarization. While 5-HT decreased isolated AMPA/kainate and NMDA receptor-mediated responses as well as slow IPSPs, we could not find a consistent effect on isolated fast IPSPs. Since in approximately 25% of subicular neurons EPSPs and slow IPSPs were reduced without any increase of membrane conductance, we conclude that 5-HT has in addition to membrane effects also effects on synaptic currents.

Animals↗
PubMed1997

Systemic administration of the phencyclidine compound MK-801 affects stimulus-induced field potentials selectively in layer III of rat medial entorhinal cortex.

Phencyclidine and related compounds such as MK-801 produce psychotic symptoms, which closely resemble schizophrenia. MK-801 causes lesions in different corticolimbic regions including the medial entorhinal cortex (mEC). Using electrophysiological recordings in brain slices we tested whether several hours of systemic administration of MK-801 affect stimulus-induced field potentials (FPs) in the mEC. Stimulus-induced FPs were selectively reduced in layer III, but not in layers II and V of the mEC. In contrast, MK-801 applied acutely over the bath in low concentration had no significant effect on evoked FPs. Since the principal cells of layer III project directly to area CA1 and the subiculum, the selective effects of MK-801 may have implications for the transfer of information to the hippocampus.

Animals↗
PubMed1997

Antioxidative and clinical effects of high-dose N-acetylcysteine in fibrosing alveolitis. Adjunctive therapy to maintenance immunosuppression.

In fibrosing alveolitis (FA), activated phagocytes cause excessive oxidative stress in the lower respiratory tract. Additionally, levels of glutathione, a major antioxidant of the human lung, are markedly reduced. Since N-acetylcysteine (NAC) is a known precursor for glutathione synthesis, we investigated the effect of NAC on redox balance and lung function in FA. Eighteen patients with an established diagnosis of FA were treated with 600 mg NAC three times daily for 12 wk in addition to their latest immunosuppressive therapy. Before and after NAC therapy, pulmonary function tests (PFTs) and bronchoalveolar lavage (BAL) were performed. BAL fluid was analyzed with regard to cell differential, glutathione status, and methionine sulfoxide content of BAL proteins (Met(O)), as an indicator of oxidative stress at the alveolar surface. There was an increase of total glutathione (GSHt = GSH +/- 2 x GSSG: 3.43 +/- 0.30 microM versus 4.20 +/- 0.66 microM, p < 0.05) and of reduced glutathione (GSH: 2.58 +/- 0.24 microM versus 3.42 +/- 0.54 microM, p < 0.005) in native BAL fluid and in the epithelial lining fluid (GSHt: 267.3 +/- 26.0 microM versus 367.1 +/- 36.0 microM, p < 0.005; GSH: 204.5 +/- 20.7 microM versus 302.9 +/- 32.2 microM, p < 0.005). The increase of GSH was accompanied by a decrease of Met(O) (6.83 +/- 0.71% versus 4.60 +/- 0.40%, p < 0.005). PFTs significantly improved during NAC treatment. We conclude that high-dose NAC significantly improved the antioxidant screen of the lungs by elevating GSH levels. Moreover, the decrease of Met(O) levels indicated an antioxidant effect at the alveolar surface. These biochemical changes were accompanied by an improvement of PFTs in patients under maintenance immunosuppression. NAC supplementation should, therefore, be considered as an adjunct therapy for FA.

Acetylcysteine↗
PubMed1997

Comparative loss of activity of recombinant secretory leukoprotease inhibitor and alpha 1-protease inhibitor caused by different forms of oxidative stress.

Secretory leukoprotease inhibitor (SLPI) and alpha 1-protease inhibitor (alpha 1-PI) are powerful antiproteases currently under investigation for their potential to protect the lung from neutrophil elastase (NE). The aim of this study was to determine whether the recombinant form of SLPI (rSLPI) and alpha 1-PI show different grades of loss of inhibitory activity when exposed to reactive oxygen metabolites. We incubated rSLPI and alpha 1-PI with N-chlorosuccinimide (NCS), chloramines, activated polymorphonuclear leucocytes (PMNs) and activated alveolar macrophages (AMs). Under all conditions evaluated, both antiproteases were partially inactivated. The resulting anti-NE activity of rSLPI was not significantly different from that of alpha 1-PI after exposure to NCS (p > 0.5), chloramines (p > 0.6), activated PMNs (p > 0.07) and activated AMs (p > 0.9). In conclusion, recombinant secretory leukoprotease inhibitor and alpha 1-protease inhibitor lose antineutrophil elastase activity to a similar extent when exposed to conditions that may be present in inflammatory lung disorders.

Animals↗
PubMed1997

Interspecies comparison of rat and hamster alveolar macrophage antioxidative and oxidative capacity.

Generation of oxidants has been implicated in lung injury and disease caused by a variety of inhaled agents such as ozone, particles, and mineral fibers. Antioxidants in the pulmonary system presumably provide the initial defense against such oxidants. We designed the present study to assess the oxidative and antioxidative capacity of alveolar macrophages (AM) from rats and hamsters. These two laboratory animal species commonly used in biomedical research are well known for their disparate response to pulmonary irritants/toxicants. AM from CD rats and Syrian golden hamsters were obtained by bronchoalveolar lavage. We assessed AM antioxidant levels by measuring the catalase and superoxide dismutase (SOD) activity and the intracellular concentrations of total glutathione, ascorbic acid, and alpha-tocopherol. We determined the AM oxidative capacity by assessing the ability of AM to oxidize extracellular glutathione (GSH) and to release superoxide anions. There were no significant differences in the intracellular antioxidant levels, except for catalase activity that was significantly (p < 0.05) higher in hamster AM than in rat AM. However, AM oxidative capacity was markedly different between the two species studied. The amount of spontaneous and phorbol myristate acetate (PMA)-induced GSH oxidation was about 5-fold higher in rat AM than in hamster AM, whereas the PMA-induced superoxide anion release did not differ significantly between the two rodents. In summary, our data suggest that species variation exists between the oxidative capacity of rat and that of hamster AM. Whereas the oxidative capacity of hamster AM appears to be based mainly on the formation of reactive oxygen species, it is suggested that rat AM possess an additional oxidative system.

Animals↗
PubMed1997

Cell size of alveolar macrophages: an interspecies comparison.

Alveolar macrophages (AM) play a critical role in the removal of inhaled particles or fibers from the lung. Species differences in AM size may affect the number and size range of particles/fibers that can be actually phagocytized and cleared by AM. The purpose of this study was to compare the cell size of rat, hamster, monkey, and human AM by selective flow cytometric analysis of cell volume. Resident AM from CD rats, Syrian golden hamsters, cynomolgus monkeys, and nonsmoking, healthy human volunteers were harvested by standard bronchoalveolar lavage procedures. Morphometric analysis of AM was performed using a flow cytometer that generates volume signals based on the Coulter-type measurement of electrical resistance. We found that hamster and rat AM had diameters of 13.6 +/- 0.4 microns (n = 8) and 13.1 +/- 0.2 microns (n = 12), respectively. Comparatively, the AM from monkeys (15.3 +/- 0.5 microns, n = 7) and human volunteers (21.2 +/- 0.3 microns, n = 10) were larger than those from rats and hamsters. The AM from humans were significantly larger (p < 0.05) than those from all other species studied, corresponding to a 4-fold larger cell volume of human AM (4990 +/- 174 microns 3) compared to hamster (1328 +/- 123 microns 3) and rat (1166 +/- 42 microns 3) AM. In summary, we have found marked species differences in the cell size of AM. We suggest that the number and size range of particles/fibers that can be phagocytized and cleared by AM may differ among species due to inherent or acquired species differences in AM cell size.

Adult↗
PubMed1996

Electrophysiological properties of rat subicular neurons in vitro.

The electrophysiological properties of 46 bursting cells and 39 regular firing cells were studied in the subiculum of rat combined hippocampal-entorhinal cortex slices. In bursting cells we found a significantly higher resting membrane potential than in regular firing cells. Upon hyperpolarization both cell types expressed a delayed inward rectification with a subsequent afterdepolarization. While in regular firing cells longer lasting depolarizing current injection caused a train of action potentials with a rather marked decline of discharge frequency, bursting cells displayed only little frequency accommodation. Regular firing cells usually displayed a fast and a slow afterhyperpolarization following a train of action potentials, while bursting neurons present only a slow afterhyperpolarization.

Animals↗