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Biomedical subjects

M Wiemann

Publications and source records attributed to M Wiemann.

At least 37 records · Page 2Linked to original sources
PubMed1998

Microcutting of living brain slices by a pulsed ultrafine water jet which allows simultaneous electrophysiological recordings (micromingotome).

Up to now microsurgical dissections in living nervous tissue (e.g. in slices or cell cultures) are performed either by micro-scalpels or by laser beams. As an alternative technique, a device for cutting with an ultrafine pulsed water jet was developed to allow precise, visually controled dissections in neuronal circuits even during electrophysiological recordings. Water is ejected by pressure (20-30 bar) from patch pipettes with tip diameters of 10-12 microm. By means of a piezo-element the pipette and the water jet are forced to oscillate vertically with a frequency of 200-400 Hz with an adjustable amplitude. These oscillations facilitate the transsection of neuronal connections even in thick slice preparations. Best results were obtained when the tip of the pipette was about 500 microm above the surface of the submerged slice tissue. This micromingotome offers the following advantages: (i) histological studies show that the water jet cleans the cutting surface, thus avoiding debris and its uncontrolable effects on cells underneath; (ii) the arrangement enables ongoing electrophysiological recordings from hippocampal slices during the cutting procedure and thus facilitates studies of the functions of neuronal connections; (iii) the device allows even disconnection in cultured nervous tissue overgrowing polyamid grids with 50 microm wide meshes.

Animals↗
PubMed1998

CO2/HCO3(-)-withdrawal from the bath medium of hippocampal slices: biphasic effect on intracellular pH and bioelectric activity of CA3-neurons.

Many studies analyzing interactions of pH and bioelectric activity focus on changes of the extracellular pH, whereas data concerning central neuronal excitability and intracellular pH (pHi) are rare. Here, we report on the spontaneous bioelectric activity and epileptiform activity of CA3-neurons during a procedure which changed pHi. As monitored in BCECF-AM loaded cells, the change from a CO2/HCO3(-)-buffered to a HEPES-buffered medium (CO2/HCO3(-)-withdrawal, hereafter termed W) was associated with a transient intracellular alkalosis (delta pH = 0.2 +/- 0.04) which preceded a sustained intracellular acidosis (delta pH = 0.4 +/- 0.04). Coinciding with this W-induced biphasic shift of pHi a biphasic alteration of spontaneous bioelectric activity was recorded: as a rule, an up to 30 min lasting increase (excitatory phase) preceded a typical sustained suppression (inhibitory phase). This biphasic action was also observed using various in vitro-epilepsy-models (bicuculline, penicillin, caffeine): epileptiform discharges were completely suppressed after an initial increase in frequency. This modulation of bioelectric activity was unlikely due to alterations of the postsynaptic GABA-system as hyperpolarizing GABAA- and GABAB-responses of CA3-neurons were hardly affected. In the majority of the neurons, the initial increase of spontaneous bioelectric activity (excitatory phase) culminated in transient burst periods lasting 5-30 min. These transient burst periods were blocked by NMDA- or AMPA-antagonists: DL-2-amino-5-phosphonovalerate (APV, 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM). The calcium-antagonist verapamil (50 microM) reduced amplitudes of depolarizations and duration of the transient burst periods. The results suggest that the biphasic alteration of pHi modulates the susceptibility of glutamate receptors and voltage-gated calcium-channels, which leads to respective changes of bioelectric activity.

Animals↗
PubMed1998

CO2-sensitive medullary neurons: activation by intracellular acidification.

Bioelectric activity of CO2-sensitive, ventrolateral medullary neurons (VLN(CS)) in organotypic cultures from the obex level of newborn rats was tested during changes of the intracellular pH (pHi) measured in BCECF-AM loaded cultures. Hypercapnia (pCO2 80-100 mmHg) reduced pHi by 0.15 +/- 0.06 units and stimulated neuronal discharges. Replacement of CO2/HCO3- in the bath by HEPES (26 mM, pH 7.4) for 10 min acidified pHi (0.07 +/- 0.03 units) and also excited VLN(CS). Ammonium chloride (10 mM, 1 min) initially alkalized (0.1 +/- 0.04) and thereafter acidified pHi (0.06 +/- 0.03), while the extracellular pH was first acidified and then alkalized. This resulted in neuronal discharge which were first suppressed and then accelerated. The findings strongly suggest that intracellular rather than extracellular acidification activates CO2-sensitive neurons.

Acidosis↗
PubMed1998

Effects of lead, mercury, and methyl mercury on gap junctions and [Ca2+]i in bone cells.

Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca2+]i). Since heavy metals compete with Ca2+ at multiple sites and increased [Ca2+]i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca2+]i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb(e) (5 mumol/liter; n = 3) and Hg(e) (5 mumol/liter; n = 3) as well as Pb(i) (25 mumol/liter; n = 7) did not change the coupling (delta MP2/delta MP1). In contrast, MeHg(e) (1-10 mumol/liter; n = 6) and Hg(i) (> or = 5 mumol/liter; n = 8) reduced the coupling to 79.5 +/- 19.3% and 62.4 +/- 15.3%, respectively, within 15-20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb(e) (5 mumol/liter, n = 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca2+]i increase. Hg(e) (n = 48) slightly increased [Ca2+]i from 100 to < or = 200 nmol/liter, whereas MeHg(e) (5 mumol/liter, n = 52) released Ca2+ from internal stores, thus increasing [Ca2+]i up to 2 mumol/liter. In conclusion, Pb(e), Pb(i) and Hg(e) do not affect gap junctional coupling per se. Since MeHg(e) and Hg(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca2+]i, which affects gap junction channels.

Animals↗
PubMed1998

A calcium release activated calcium influx in primary cultures of rat osteoblast-like cells.

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca2+ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca2+]e) by 5 microM thapsigargin (TG) or 2 microM A23187. Readdition of 1.8 mM [Ca2+]e increased the free intracellular Ca2+ ([Ca2+]i) after a delay of 30-60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca2+]e and was substantially lowered if readdition of 1.8 mM [Ca2+]e was preceded by, e.g., 0.72 mM [Ca2+]e. CRAC-induced [Ca2+]i peaks were correlated (r = 0.543) with [Ca2+]i peaks during the complete depletion of ICS with A23187. Ca2+ influx due to CRAC could be blocked by flufenamic acid (100 microM) but not verapamil (20 microM). Ni2+ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca2+]i influx rate. Induction of CRAC enhanced the influx of Mn2+ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca2+. This Ca2+ flux may be activated by transmembraneous gradients of Ca2+ and Ni2+.

Animals↗
PubMed1997

Epileptic neurons induce augmenting synaptic depolarizations in non-epileptic neurons (buccal ganglia, Helix pomatia).

Spread of epileptic activity was studied by inducing epileptiform activity (pentylenetetrazol, PTZ) in one part of a nervous system and by analyzing responses of neurons in a non-PTZ-treated part (identified neurons, paired buccal ganglia, Helix pomatia). Paroxysmal depolarization shifts (PDS) induced time-locked depolarizations in non-epileptic neurons (latency ca. 5 s, duration ca. 1 min, amplitude < or =20 mV). Amplitudes were augmenting during several hours of epileptic activity. Depolarizations were accompanied by an increase in membrane resistance and they were blocked in 'high Mg-low Ca' solutions. It is assumed that the potentials represent a typical widespread response of non-epileptic neurons to PDS of other neurons. This response may be induced via non-specific releases of substances of the epileptically active neurons thereby activating neighboring neurons which in turn activate neurons in control ganglion.

Animals↗
PubMed1996

Simultaneous blockade of intracellular calcium increases and of neuronal epileptiform depolarizations by verapamil.

The specific L-type calcium channel blocker verapamil exerts an antiepileptic effect on neurons. This effect is assumed to depend on the blockade of transmembraneous calcium flux during epileptic discharges. In order to test this hypothesis, fura-dextran loaded snail neurons were rendered epileptic by pentylenetetrazole (40 mmol/l). The effect of verapamil (20 or 40 mumol/l) on free intracellular calcium ([Ca2+]i) transients was investigated by means of fluorescence ratio-imaging and simultaneous intracellular membrane potential recording. During epileptic depolarization [Ca2+]i increased especially in the outermost submembraneous areas of the neuron. [Ca2+]i reached peak values 6-22 s after the onset of epileptic depolarizations. Application of verapamil progressively shortened the epileptic depolarizations. This shortening of epileptic depolarizations developed along with a diminution of the submembraneous calcium signals down to noise level. The effect was found to be reversible. It is concluded that the antiepileptic effect of verapamil depends largely on its ability to block transmembraneous calcium flux.

Animals↗
PubMed1996

Effects of calcium on gap junctions between osteoblast-like cells in culture.

The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca2+]i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca2+]i, patch pipette solutions contained 100 micromol/liter Ca2+. About 1-2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca2+ to 20 in high Ca2+. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca2+ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca2+]i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca2+]i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na+-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca2+]i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca2+.

Animals↗
PubMed1996

Alterations of neuronal fibers after epileptic activity induced by pentylenetetrazole: fine structure investigated by calcium cytochemistry and neurobiotin labeling (buccal ganglia, Helix pomatia).

The influence of epileptic activity on both the fine structure of neuronal processes and the subcellular distribution of calcium-binding sites was investigated in an epileptic model system, the buccal ganglion of Helix pomatia. Pentylenetetrazole was used to induce epileptic activity. Calcium-binding sites were visualized as electron-dense precipitates. Epileptic and control activity was intracellularly recorded from neuron B3 labeled with neurobiotin. After epileptic treatment, many processes contained vacuolated or electron-lucent areas next to morphologically intact areas. Most of these areas were enveloped by layers of endoplasmic reticulum. Lamellar formations of membranes occurred frequently. Calcium cytochemistry revealed a high content of dense precipitates within these formations of the endoplasmic reticulum. Local accumulations of diffuse precipitates were more frequent after epileptic activity than in controls. In contrast, structures such as lamellar bodies, cytosomes, and synapse-like formations, all of which contained many electron-dense precipitates, were apparently unchanged after epileptic activity. This study demonstrates that epileptic activity can lead to local degeneration of neuronal fibers and an associated increase in calcium-binding sites. It is suggested that calcium sequestration is locally increased within neuronal processes during epileptic activity.

Animals↗
PubMed1996

A method to study the effects of an epileptic focus on non-epileptic nervous tissue.

Locally applied epileptogenic drugs directly affect the local cells which project their epileptic activity into the surrounding tissue. This paper describes an experimental set-up which allows differentiation of direct effects of an epileptogenic drug from indirect ones, which are synaptically projected. The set-up consisted of an experimental chamber which enabled the isolated superfusion of a part of a nervous system with a drug. Isolated superfusion was obtained by means of a divider between two compartments of the experimental chamber and of an outlet for the bath solutions below the divider. Quality of isolation was tested with unilateral application of the epileptogenic drug pentylenetetrazole (PTZ) and of the dye methylene blue. Distribution of extraganglionic and intraganglionic PTZ was measured by PTZ-selective microelectrodes. It was found that the drug could not be detected at the non-drug side. Correspondingly, unilateral dye application resulted in a sharp border between the treated and untreated side. The experimental set-up was used to study projected events evoked by sustained epileptic activity of defined parts of a nervous system. It was found that the non-treated cells became progressively coupled to the epileptic neurons in the course of the experiments.

Animals↗
PubMed1996

Impaired stem cell collection by consecutive courses of high-dose mobilizing chemotherapy using cyclophosphamide, etoposide, and G-CSF.

Tandem cycles of myeloablative chemotherapy can increase dose intensity and total dose of chemotherapy, but sufficient numbers of progenitor cells must be collected to ensure hematologic recovery after each treatment. This study was undertaken to determine if two courses of mobilizing chemotherapy given 4 weeks apart using cyclophosphamide 4000 mg/m2 and etoposide 400 mg/m2, combined with G-CSF 5-10 mg/kg on days 3-16 could each provide sufficient numbers of peripheral blood progenitor cells to support tandem cycles of myeloablative chemotherapy in 20 patients with stage IV breast cancer. Leukapheresis of blood with WBC > 1000/mm3 was performed daily for up to five collections (days 12-16), and mononuclear cells, CFU-GM, and CD34+ cells were compared between the first and second collections. The second course of mobilizing treatment resulted in similar numbers of mononuclear cells collected but far fewer CFU-GM and CD34+ progenitor cells. This prevented using the second collection of progenitor cells as the sole source for the second transplant. The data suggest that a second course of cyclophosphamide, etoposide, and G-CSF given 4 weeks after the first leads to progenitor cell depletion, and efforts to increase the yield of blood-derived progenitors should focus on the initial mobilizing procedure.

Adult↗
PubMed1996

Thymic nurse cells: entry of exogenous horseradish peroxidase via a transcytotic route.

In order to address the question whether thymocytes inside thymic nurse cells (TNCs) are secluded against exogenous proteins, TNCs were isolated and kept in the presence of horseradish peroxidase. The distribution of horseradish peroxidase reaction product (HRP) within the TNCs was studied by light (LM) and electron microscopy (EM). Stained and unstained TNCs could be distinguished by LM. At the EM level, stained TNCs had a widened lumen and HRP lined the luminal surfaces. In unstained TNCs the lumen appeared normal and contained almost no HRP. In both types of TNCs the epithelial cell contained HRP in endosomes and, together with cellular debris, in larger vacuoles. Exocytosis of the latter was observed. Conglomerates of HRP were found inside the lumen and also within thymocytes. Data suggested that TNCs can shield secluded thymocytes against exogenous proteins except those which are provided by a transcytotic pathway. Such a pathway has not been demonstrated before in TNCs.

Animals↗
PubMed1995

Epileptic discharges induced by pentylenetetrazol: ultrastructural alterations in identified neurons and glial cells (Helix pomatia).

The effects of sustained epileptic activity induced by pentylenetetrazol on morphology of buccal ganglia of Helix pomatia were investigated. Neuronal somata and processes as well as glial cells were evaluated after 5 hours of epileptic activity and after 5 hours under control conditions. After epileptic activity neurons showed signs of degeneration consisting of condensation of nuclear chromatin, decreased activity of Golgi apparatus, increased numbers of lamellar bodies and multivesicular bodies, clusters of vesicles and vacuoles, loss of microtubuli, and scattered lamellar bodies. Neuronal somata and large neuronal processes appeared less affected than the smaller processes. Glial cells showed signs of phagocytotic activity as increased cell size, numerous degenerating neuronal processes within the cytoplasm as well as lysosome like bodies and vacuoles. The changes developing along with epileptic activity were interpreted to indicate degeneration and subsequent phagocytotic activity of neuronal processes in synaptic regions of the ganglia. Thus, evidence is presented for synaptically induced degenerative processes in an intact nervous tissue that is not affected by seizure-induced alterations of respiration or systemic circulation.

Animals↗
PubMed1995

Interferon alpha-2a and external beam radiotherapy in the initial management of patients with glioma: a pilot study of the National Biotherapy Study Group.

The National Biotherapy Study Group conducted a phase I/II trial of alpha-interferon (IFN) plus radiation therapy (RT) in glioma patients to confirm the feasibility of combining these two modalities. Patients newly diagnosed gliomasreceived external beam RT as 180 cGy in 33 fractions over six to seven weeks, five days a week, and IFN at a dose of 3 MIU SC Monday, Wednesday and Friday of each week. IFN was increased to 5 MIU after two weeks and was given for up to 16 weeks. Patients were monitored for toxicity and failure-free and overall survival. There were 12 men and seven women with an age range of 24-77, and a median age of 64 years. There were 12 glioblastomas and seven advanced astrocytomas. Complete surgical resection was carried out in two patients, nine had a partial resection, and eight had a biopsy only. Two patients in the latter group deteriorated rapidly and received < 2 weeks of RT/IFN. One patient stopped IFN because of a skin rash, another stopped because of concurrent pneumonia, and one patient was noncompliant. RT and IFN were well-tolerated; 14 of the 19 patients completed the eight weeks of IFN/RT. However, only three patients took IFN for the maximum of 16 weeks. The only grade 4 toxicities noted were increases SGOT in three, increases alk phos in two, and severe fatigue in four patients. The median failure-free survival was two months, median survival was 7.5 months, and four patients survived beyond one year. The longest survivor was 29.1 months, and one patient is still alive after 20.7 months. IFN/RT can be safely co-administered in patients with gliomas. A randomized trial would be needed to establish clinical benefit.

Adult↗
PubMed1993

Subcellular localization of immunoreactive oxytocin within thymic epithelial cells of the male mouse.

Immunoreactive oxytocin is expressed by thymic epithelial cells, which share properties with neuroendocrine cells. In order to investigate the assumed paracrine secretion of oxytocin, we studied the subcellular localization of immunoreactive oxytocin within thymic tissue and cultured thymic epithelial cells of the male mouse. Three types of immunoreactive cells were distinguished with the electron microscope. Immunoreactive oxytocin was found to be restricted to the cytoplasm by the use of pre- and postembedding methods. Some epithelial cells, especially in the cortex, showed a pronounced labelling of vesicular membranes and membrane tubules of the endoplasmic reticulum. In some cells, keratin filaments were associated with the electron-dense stain. Under culture conditions immunoreactive cells of different shapes were found, all displaying similar patterns of labelling. The contents of different types of vacuoles were only rarely labelled. A special class of immunoreactive exocytotic vesicles could not be identified. Thus, our results do not support neuroendocrine secretion of oxytocin via vesicles of thymic epithelial cells but offer alternative modes of secretion.

Animals↗
PubMed1993

Effect of oxytocin on free intracellular Ca2+ levels and progesterone release by human granulosa-lutein cells.

Oxytocin and its receptor are found in the corpus luteum in a variety of species, including the human. In the present study we used fura-2 microfluorimetry to investigate whether activation of the oxytocin receptor of cultured human granulosa-lutein cells causes intracellular calcium (Ca2+) signals and affects progesterone release. Although after 1 day in culture, cells were not responsive to oxytocin, the number of responsive cells increased steadily during the first 3 days in culture, reaching a maximum on days 4 and 5 (59-66%) and then declined again until day 8. Effective oxytocin concentrations were apparently independent of the culture day, and concentrations as low as 10 nmol/L increased intracellular free Ca2+ levels from 70-140 nmol/L (basal levels) to maximal peak levels of 800 nmol/L. The oxytocin-induced Ca2+ signal was not affected by removal of extracellular Ca2+ with EGTA. Moreover, depletion of intracellular Ca2+ stores by ionomycin treatment rendered the cells unresponsive to oxytocin, pointing also at the intracellular source of the oxytocin-inducible Ca2+ signal. Interestingly, after one single stimulation with oxytocin, cells became refractory to additional stimuli, and only extremely high concentrations of oxytocin induced a second increase in intracellular free Ca2+. To examine the possible effects of oxytocin on progesterone release by cultured cells, we incubated cells on culture day 2 (20% responsive cells in the fura measurements) and culture day 5 (66% responsive cells in the fura measurements) for 24 h with oxytocin (10 nmol/L) and hCG (10,000 IU/L). Although hCG significantly stimulated progesterone release, oxytocin alone was without a stimulatory effect on either day. However, a significant augmentation of the effect of hCG on progesterone release was found in incubations of cells on day 5. Interestingly, the effects of hCG also included stimulation of oxytocin release by cultured granulosa-lutein cells into the culture medium, as determined by RIA. In summary, our data indicate the presence of a functional oxytocin receptor on human granulosa-lutein cells that is linked to Ca2+ as a second messenger released from intracellular Ca2+ stores. The number of oxytocin-responsive cells increases during differentiation in culture. Moreover, oxytocin release induced by hCG and a stimulatory effect of oxytocin on the hCG-induced progesterone production during the period of maximal responsiveness of cultured cells were found. We, therefore, propose that oxytocin may have autocrine and/or paracrine functions in human granulosa-lutein cells, including fine-tuning of progesterone release.

Calcium↗
PubMed1991

Identification and localization of major stage-specific polypeptides of infectious Holospora obtusa with monoclonal antibodies.

With the help of monoclonal antibodies (MAbs) we investigated the occurrence of six polypeptides throughout parts of the life cycle of Holospora obtusa, a bacterium infecting the macronucleus of the ciliate Paramecium caudatum. The polypeptides of interest formed major bands in the protein pattern of the infectious form (IF) of H. obtusa. All MAbs used recognized individual polypeptide bands of the IF proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides were also detected in the reproductive form in trace amounts. Two-dimensional electrophoresis revealed that the 33,000-, 28,000-, and 14,000-Mr polypeptides wre acidic and exhibited multiple isoelectric points under native conditions. Four polypeptides (Mrs of 50,000, 33,000, 28,000, and 20,000) were no longer detected or became drastically reduced within the first 30 min of invasion. Concomitantly, a loss of electron-dense periplasmic material occurred, which is typical for invading IFs (H.-D. Görtz and M. Wiemann, J. Protistol. 24:101-109, 1989). In an attempt to clarify the subcellular localization of the six polypeptides, we performed chloroform extraction studies and identified four of the released polypeptides with MAbs. A 14,000-Mr polypeptide was immunocytochemically localized in the periplasm of the IF. The results showed that the six major polypeptides of the IF were stage specific or stage specifically enriched and are likely to contribute to the electron-dense periplasmic material of the IF.

Animals↗