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At least 343 records · Page 19Linked to original sources
PubMed1996

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants.

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.

Acetylcholine↗
PubMed1994

The uncoupling effect of diacylglycerol on gap junctional communication of mammalian heart cells is independent of protein kinase C.

Possible regulatory effects on cell-to-cell communication of a synthetic diacylglycerol, an activator of protein kinase C (PKC), were examined in pairs of synchronously beating ventricular myocytes of neonatal rats in primary culture. Junctional communication was estimated by measuring either the rate constant of dye diffusion, with the fluorescence recovery after photobleaching technique, or the cell-to-cell electrical conductance with a double whole-cell voltage clamp. The addition of a freshly prepared emulsion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 micrograms/ml), either in the bath or in the solution filling the patch pipet, was seen to interrupt intercellular communication within approximately 8 to 10 min. This effect is neither mimicked by stimulation of PKC by a phorbol ester, nor prevented by PKC inhibitors, making it unlikely that, in these cells, PKC activation could induce intercellular uncoupling. During OAG exposures, the intracellular calcium concentration was very modestly increased (by a factor 1.5 to 2), which does not suffice to account for uncoupling. OAG might trigger interruption of cell-to-cell communication by a mechanism analogous to that of other lipophilic molecules (such as aliphatic alcohols or long chain unsaturated fatty acids) which interfere with gap junctions.

Animals↗
PubMed1994

Dual-color detection of DNA sequence variants by ligase-mediated analysis.

Genetic screening for sequence variants associated with disease is assuming increasing importance in clinical medicine as well as in research. We describe an efficient method for such analyses, comprising a combination of practical features: (1) Amplified DNA samples are analyzed for their ability to serve as templates in standardized allele-specific ligation reactions between oligonucleotide probes; (2) Two allele-specific probes, differentially labeled with either of two lanthanide labels, compete for ligation to a third oligonucleotide (the signal from the two labeled probes can thus be directly compared in a sensitive time-resolved fluorescence detection reaction); and (3) Large sets of analyses are processed in parallel using a 96-pin capture manifold, serving to reduce pipetting steps and the risk of contamination. We present here the basis of the technique and its application to the screening for two common mutations causing cystic fibrosis and alpha 1-antiytrypsin deficiency.

Base Sequence↗
PubMed1999

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project.

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.

Algorithms↗
PubMed1993

Experiences on the application of the polymerase chain reaction in a diagnostic laboratory.

Double polymerase chain reaction (PCR) assays with nested primers have been applied in a routine laboratory for the diagnosis of herpes-, pesti- and retroviral infections of animals. Various methods and tools have been tested to prevent and to eliminate false positive results as well as to visualize the PCR products (amplicons). The u.v. and DNase treatments proved to be unsuitable for decontamination of PCR mixtures contaminated with amplicons shorter than 380 bp. By constructing special tube-holders and openers, and by applying a simple technique of pipetting, the false-positive PCR results were eliminated. The PCR products were visualized by three simple methods. The solid phase colorimetric method termed 'Detect Immobilized Amplified DNA' (DIANA) has been adapted to microplate. The other method, termed 'Colorimetric Detection Assay on Filter' (CODAF), proved to be very rapid. However, despite these advantages of DIANA and CODAF, henceforward the nucleic acid hybridization methods were found most reliable for safe identification of PCR amplicons. In order to simplify the hybridization, various non-radioactive labelling methods of oligonucleotide probes were compared. Biotinylation at the 5' end by means of oligonucleotide synthesis was the most simple and practical labelling method in this laboratory. The routine applicability of hybridization was further simplified by constructing a robot device, which automatically performs filter-hybridization and subsequently develops the signals derived from the biotinylated hybrids.

Clinical Laboratory Techniques↗
PubMed1996

The use of mimics as internal standards to avoid false negatives in diagnostic PCR.

The PCR laboratories may face not only the frequently documented false positive results, but also unexpected false negatives. The latter are mostly due to inhibitory effects of some ingredients and/ or to pipetting errors. In order to reveal the errors, it is advisable to apply standard molecules as indicators of the efficacy of the reactions. In the present paper a rapid and simple method is presented to create internal standards for two test PCR assays. One of the assays detects proviral DNA of bovine leukemia virus (BLV-PCR), the other assay amplifies cDNA of feline infectious peritonitis virus (FIPV-RT-PCR). The internal standard molecules, termed 'mimics', were constructed to have the same primer-binding nucleotide sequences as the viral nucleic acids, but to flank a heterologous DNA fragment of different size. As heterologous DNA, a part of human beta-acin gene was used for the mimic construction. The identical primer-binding nucleotide sequences allowed co-amplification of the viral nucleic acid and the mimic in the same tube, and simultaneously, the size differences allowed easy discrimination between their PCR products. By running a rapid agarose gel electrophoresis after co-amplification, the presence or absence of the mimic PCR products provided proper information on the efficacy of the PCR in each reaction tube. We came to the conclusion that 5 to 20 mimic molecules, co-amplified with the samples, significantly increased the reliability of the diagnostic PCR assays.

Base Sequence↗
PubMed1996

Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat.

4-Vinylcyclohexene diepoxide (VCD) destroys small preantral (25-100 microns) ovarian follicles after repeated dosing in mice and rats. A previous study determined this follicular destruction is via apoptosis (physiological cell death). The purposes of this study were to examine the effects of VCD on amounts of mRNA for several genes that might be involved in this ovotoxic response and to determine the specificity of this response for small preantral follicles. The genes of interest were bax, a cell death gene; three forms of the antioxidant enzyme, superoxide dismutase (mitochondrial manganese-containing or MnSOD, cytosolic copper/zinc-containing or Cu/ZnSOD, and secreted or secSOD); and microsomal epoxide hydrolase (mEH), involved in detoxification of VCD. Female Fischer 344 rats were administered daily doses (10 days) of vehicle control (sesame oil) or VCD (80 mg/kg, ip). Four hours after the last injection, livers and ovaries were removed. Small (25-100 microns) and large (100-250 microns) preantral follicles were separated from the ovaries by gentle dissociation and collected by mouth pipeting. Total RNA was extracted from all tissues, reverse transcribed into first-strand cDNA, and amplified by polymerase chain reaction using oligonucleotide primers specific for each gene. Relative levels of mRNA were visualized by agarose gel electrophoresis and autoradiography and quantified by densitometric analysis. Coamplification of ribosomal protein L19 (constitutively expressed in ovarian tissue) was used for normalization in each sample. Increased levels of mRNA for bax (172 +/- 20% of control, p < 0.05), MnSOD (248 +/- 70% of control, p < 0.05), and mEH (352 +/- 120% of control, p < 0.05) were measured in 25- to 100-microns follicles collected from VCD-treated compared with control rats. Unlike 25- to 100-microns follicles (the targets of ovotoxicity), in 100- to 250-microns follicles (nontargets) there were no changes (p > 0.05) in mRNA levels for bax or MnSOD in VCD-treated rats; however, mRNA levels for mEH were significantly decreased (79 +/- 4% of control, p < 0.05), compared with control. No changes in levels of mRNA for mEH were observed in liver from VCD-treated rats relative to control. Additionally, in liver VCD caused a significant decrease in mRNA levels for bax (31 +/- 5% of control, p < 0.05) and Cu-ZnSOD (56 +/- 17% of control, p < 0.05). In summary, dosing of rats with VCD enhanced expression of mRNA encoding several genes that might respond during the induction of ovotoxicity. The selective increase in bax in the population of follicles destroyed by repeated dosing with VCD may reflect their susceptibility to apoptosis.

Animals↗
PubMed1979

Studies of isolated and enriched rat antral mucosa gastrin cells.

A technique has been developed to obtain viable, isolated and enriched populations of gastrin cells (G-cells) from the rat stomach. Restricted tissue samples from a small area of the pyloric antrum known to be particularly rich in G-cells, were sequentially digested with pronase followed by mechanical agitation, to remove the epithelial cells. This technique resulted in a significant enrichment of G-cells (3-4 fold) since the surface epithelial cells and upper portions of the glands were discarded before the initial G-cell fraction was collected. These cells in suspension were then isolated from each other by gentle pipetting in a DNase containing solution and designated the crude preparation (CP). The G-cells were then purified further by separating the cells according to size by velocity sedimentation. The greatest concentration of G-cells (15-25%) was found in the fraction containing cells with diameters of 10 to 12 micrometer. The effectiveness of the technique was evaluated by counting G-cells as identified by electron microscopy and immunofluorescence and assessing gastrin activity by radioimmunoassay. All three methods indicated that cell separation by gravity velocity sedimentation enriched the G-cell population 15-20 fold over their concentration in the CP. The combined techniques of selective pronase digestion followed by gravity velocity sedimentation resulted in an isolated cell preparation containing a 50-100 fold increase of G-cells over their normal distribution in the intact gastric mucosa. Since these isolated G-cells retain features indicating viability, their usefulness for in vitro studies is suggested.

Animals↗
PubMed1983

Energy-dispersive X-ray fluorescence determination of platinum in plasma, urine, and cerebrospinal fluid of patients administered cis-dichlorodiammineplatinum(II).

A method involving the use of an energy-dispersive X-ray fluorescence spectrometer was developed for assaying total platinum concentrations in body fluids of patients treated with the antitumor drug cis-dichlorodiammineplatinum(II). Sample preparation by this procedure is simple, consisting in adding an internal standard (Zr) to 1 ml of biological fluid or tissue homogenate, pipetting 20 microliter of the sample onto a Mylar sample holder, and drying. This produces a thin-film sample, which effectively eliminates absorption enhancement effects due to other elements in the specimen. Standard addition studies were found to be linear in the concentration range of interest (0.1-10.0 micrograms/ml), with correlation coefficients exceeding 0.99. Minimum detection limits range from 0.10 to 0.25 micrograms Pt per ml, depending on the body fluid, which is adequate for routine patient monitoring after normal chemotherapeutic doses of cis-dichlorodiammineplatinum(II). In preliminary studies with mammalian liver, standard addition experiments were found to be linear and the minimum detection limit was found to be 1.4 micrograms/g dry weight.

Body Fluids↗
PubMed1983

An optical micromethod for the determination of relative crystallisation rates of calcium oxalate in gels: method and preliminary results.

This paper describes a new, highly efficient micromethod for the determination of relative crystallisation rates of calcium oxalate (CaOx). Crystallisation is performed in the upper layer of a gel (bactoagar, agarose) which contains one component (oxalate) of the sparingly soluble salt. Precipitation is started by pipetting Ca++ containing solutions (in the presence and absence of crystallisation inhibitors) onto the gel. The process is followed quantitatively as a function of time by means of vertical light path photometry carrying out quasi-simultaneous multideterminations within a 50-fold multicuvette. The test volume is 0.1 ml. The method is suitable for large scale determinations. About 50 single crystallisation kinetics can be measured within 5-15 min. Testing three known inhibitors of CaOx crystal formation, relative inhibitory activities were obtained with standard errors of 1%-4%.

Calcium Oxalate↗
PubMed1981

A method for the purification of single A, B and D cells and for the isolation of coupled cells from isolated rat islets.

A method has been developed for the purification of single A, B and D cells and for the isolation of coupled islet cells. Isolated rat islets were dissociated by repeated pipetting in the presence of trypsin and ethylene glycol bis (beta-aminoethyl ether) N,N' tetra-acetic acid (EGTA), and then filtered through nylon and Percoll; the cell preparation consisted of 70% single and 30% coupled cells. Sizing of the cells led to the recognition of a small-islet cell population (35%; cell volume 200-600 micrometer 3) composed of A and D cells, and a large-islet cell population (65%; cell volume 600-1500 micrometer 3) identified as B cells. Differences in sedimentation velocity formed the basis for the islet cell separation by counterflow elutriation. Single islet cells eluted prior to coupled cells and were distributed over A and D cell-enriched fractions I and II (65% A, 25% B, 10% D) and the B cell-enriched fraction III (93% B). The slightly different densities of A (d = 1.068), B (d = 1.065) and D (d = 1.070) cells allowed a subsequent purification by density gradient centrifugation resulting in a final 10- to 30-fold enrichment in either A, B or D cells. Most coupled islet cells were recovered in fraction IV, occurred mainly as doublets and were composed of 90% B cells and 7% D cells; the multiple pseudopods, which characterize isolated D cells, might contribute to the coupling tendency of the D cells. It is concluded that the purified A, B and D cell fractions and the coupled islet cell preparations offer a direct approach to the study of individual islet cell types and their intercellular communication.

Animals↗
PubMed1991

Voltage-dependent calcium current in dissociated smooth muscle cells of the buccal mass of Aplysia.

Isolated smooth muscle cells of the buccal mass of Aplysia contracted in response to depolarization elicited by a patch electrode in whole-cell configuration. With cesium-containing pipet solution and tetraethylammonium and 4-aminopyridine in the external solution depolarization elicited inward current. The voltage-dependent inward current was blocked completely by lanthanum (10 mmol.l-1), inhibited 80-90% by nifedipine (1 mumol.l-1), and was dependent upon extracellular calcium. These results showed that the voltage-dependent inward current was due to activation of voltage-dependent calcium channels (VDCaCH). Minimal depolarization to begin activating VDCaCH was -60 to -30 mV. Inward current peaked within 8 ms and then decreased rapidly to a lower level of relatively non-inactivating current. The initial peak current could be mostly inactivated by a depolarization to -20 mV for 500 ms. Nifedipine reduced both the peak current and the relatively non-inactivating current. Nifedipine inhibited high potassium-elicited contractions of both intact and dissociated muscle. These results suggested that VDCaCH mediates calcium influx which triggers contraction in molluscan smooth muscle fibers.

4-Aminopyridine↗
PubMed1983

Recovery of Ca currents from inactivation: the roles of Ca influx, membrane potential, and cellular metabolism.

Ca currents were examined with regard to their recovery from inactivation. The experiments were done on isolated nerve cell bodies of Helix aspersa using a combined suction pipet , microelectrode method for voltage clamp, and internal perfusion. Ca currents were separated by suppressing K and Na currents. The time course of recovery was determined by applying a test pulse at intervals ranging from 1 msec to 20 sec after prepulses varying from 20 to 3000 msec in duration. Each pair of pulses was preceded by a control pulse to ensure that the Ca currents had recovered before the next test pair was applied. Ba and Ca currents were compared and the effects of intracellular perfusion with EGTA, ATP, and vanadate were examined. Ba currents recovered in two stages and this time course was well fit by a sum of two exponentials with amplitudes and time constants given by A1 and tau 1 for the fast component and A2 and tau 2 for the slow component. In Ba the time constants were unchanged when prepulse durations were prolonged from 70 to 700 msec, although the initial amplitudes A1 and A2, particularly A2, were increased. Comparable influxes of Ca during the prepulse caused much more inactivation, but interestingly the recovery occurred at the same rate. The time course of Ca current recovery was also fit by a sum of two exponentials, the time constants of which were both smaller than the time constants of Ba current recovery. However, the time constants of Ca current recovery were increased markedly when prepulse durations were prolonged. Increasing the extracellular Ca concentration had a similar effect. Increasing the Ba influx had no effect on the recovery time constants, and the Ba results are consistent with reversible inactivation gating of potential-dependent membrane Ca channels. The Ca results show that Ca influx enhances inactivation. Intracellular perfusion with EGTA resulted in less inactivation in the cast of Ca but it had no effect on Ba currents. Intracellular ATP increased the rate of recovery of Ca currents, and intracellular vanadate inhibited recovery. It is concluded that recovery of Ca channels depends upon both Ca influx and membrane potential and is modulated by agents which affect Ca metabolism.

Adenosine Triphosphate↗
PubMed1994

beta-Amyloid Ca(2+)-channel hypothesis for neuronal death in Alzheimer disease.

The Alzheimer's Disease (AD) amyloid protein (A beta P[1-40]) forms cation selective channels when incorporated into planar lipid bilayers by fusion with liposomes containing the peptide. Since the peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, we also tested the possibility of direct incorporation of the soluble A beta P[1-40] into the membrane. We found the peptide can also form similar channels in acidic phospholipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome mediated incorporation, the A beta P[1-40]-channel in the solvent-free membrane patch exhibits multiple cation selectivity (Cs+ > Li+ > Ca2+ > or = K+), and sensitivity to tromethamine. The fact that equivalent A beta P[1-40] amyloid channels can be detected by two different methods thus provides additional validation of our original observation. Further studies with a beta P-channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective but loses its tromethamine sensitivity. By contrast, amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would disrupt cellular homeostasis. We therefore interpret these data as providing further support for our beta-amyloid Ca(2+)-channel hypothesis for neuronal death in Alzheimer's Disease.

Alzheimer Disease↗
PubMed1986

The application of flow cytophotometry in measurements of cell adhesion.

A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

A dissolution rate apparatus for the prediction of initial drug absorption patterns in beagles: tolbutamide tablets.

An apparatus utilizing liquid turbulence to simulate hydrodynamic conditions generated by gastrointestinal peristalsis was designed to estimate drug release from solid oral dosage forms; This turbulence was achieved through special arrangements of a pipetting pump to a dissolution chamber. By adjusting the flow rate of the pump to deliver and withdraw a fixed volume of dissolution medium per minute, a correlation was developed between dissolution rates and absorption patterns in beagles of two commerical tolbutamide tablets, A and B, and a micronized tolbutamide suspensionmon the basis of this relationship, it was possible to predict the initial absorption patterns of two misformulated tablets, C and D.

Animals↗
PubMed1990

The use of an alginic acid matrix to support in vitro development of isolated murine blastomeres.

The present study reports the use of a cross-linked calcium alginate matrix to replace the zona pellucida in supporting in vitro development of isolated two-cell murine blastomeres. The zona pellucidae were enzymatically removed with a 0.5% pronase solution and blastomeres separated with a fine-bore glass pipet. The matrix was created by coating the blastomeres with a 3.5% sodium alginic acid solution, which forms a mechanically stable complex upon transfer to media containing high concentrations of a divalent cation. Blastocyst formation was 55/61 (90.2%) for untreated intact embryos and 61/87 (70.1%) and 27/65 (41.5%) for blastomeres cultured with and without the artificial matrix respectively (P less than 0.001).

Alginates↗
PubMedDate not supplied

Layer by layer analysis of commissural hippocampal evoked potentials during glutamate microiontophoresis.

Commissural evoked potentials (EP) in hippocampal area CA1 were investigated during microiontophoretic application of sodium glutamate (SG) in experiments on unanesthetized rats immobilized with diplacin. EP were recorded by the central barrel of a five-barreled microiontophoretic microelectrode before and during application of SG. The effect of SG in the region of EP reversal was characterized by depression of the initial positive phase of the response and an increase in negativity. In the radial layer SG caused a substantial increase in the commissural response recorded at a depth. At a distance of 100 mu from the tip of the iontophoretic pipet no changes in EP were found. An increase in the intensity of transhippocampal stimulation was accompanied by a marked increase in the amplitude and a change in shape of the individual phases of EP and it abolished the effect of the microiontophoretically applied SG. During paired stimulation with an interval of 20--200 msec different effects of SG on the testing response were recorded depending on the length of the interval.

Animals↗