Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.


A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.


3' non-translated sequences in Drosophila cyclin B transcripts direct posterior pole accumulation late in oogenesis and peri-nuclear association in syncytial embryos.

We have characterised forms of the Drosophila cyclin B transcript that differ as a result of a splicing event which removes a nucleotide segment from the 3' untranslated region. In oogenesis, both cyclin A RNA and a shorter form of the cyclin B transcript are seen in the cells of the germarium that are undergoing mitosis. The shorter cyclin B transcript alone is then detectable in the presumptive oocyte until stages 7-8 of oogenesis. Both cyclin A RNA and a longer form of the cyclin B RNA are then synthesised in the nurse cells during stages 9-11, to be deposited in the oocyte during stages 11-12. These transcripts become evenly distributed throughout the oocyte cytoplasm but, in addition, those of cyclin B become concentrated at the posterior pole. Examination of the distributions of RNAs transcribed from chimeric cyclin genes indicates that sequences in the 3' untranslated region of the larger cyclin B RNA are required both for it to become concentrated at the posterior pole and to direct those transcripts in the body of the syncytial embryo to their peri-nuclear localisation. These sequences are disrupted by the splicing event which generates smaller cyclin B transcripts.


Pleomorphic adenoma: a preliminary histopathologic comparison between tumors occurring in the deep and superficial lobes of the parotid gland.

Twenty-four cases of pleomorphic adenoma of the parotid gland were retrospectively reviewed. The histopathological features of capsular thickness, penetration and completeness, tumoral cell population and mitotic activity were analyzed in twelve superficial lobe and twelve deep lobe tumors. The capsules were found to be significantly thicker and less likely to be penetrated by tumor in the deep lobe group. No significant differences in completeness of encapsulation, cell population or mitotic activity were found between the two groups. This may explain the clinical suggestion that the less aggressive surgical approach which is often performed does not compromise the prognosis for recurrence in the deep lobe lesions.


Sinusitis--inspecting the causes and treatment.

In summary, sinusitis is a common disease caused by viruses, bacteria and the accumulation of excessive secretions and inflammatory mediators that impair the function of the mucociliary transport. Combination treatment is usually necessary to treat the cause and relieve the symptoms of sinusitis. Therapy aims at eliminating causative bacteria with antibiotics, decongesting edematous membranes, and thinning mucus with use of a mucolytic-expectorant. Improving the rheology of mucus by thinning abnormally thickened secretions may improve mucociliary transport and enhance penetration of antibiotics. Acute sinusitis usually responds to treatment within 2 weeks. However, if treatment is unsuccessful or a severe complication occurs, intravenous antibiotics may be necessary along with antral puncture and lavage. In resistant cases, an appropriate surgical procedure may enhance the drainage.


Inverted papilloma of the nose and paranasal sinuses in children.

Inverted papillomas are rare nasal and sinus neoplasms in children. The disturbing biological characteristics of these tumors seen in adults, including malignant degeneration and the high frequency of recurrence, have been similarly observed in the pediatric population. Therefore, inverted papilloma in children mandates similar treatment to that proven effective in adults, including wide excision and careful histopathological examination of any tissue excised. Radiation therapy should be reserved for those tumors associated with carcinoma. Since recurrences can occur after long periods of time, life-long follow-up is warranted.


Postcricoid surface laryngeal electrode.

Progress in identification of the recurrent laryngeal nerve in thyroid and parathyroid surgery has lagged behind the identification of other head and neck nerves. Attempts to use electronic pulsed nerve stimulators with evoked response electromyography have failed due to the absence of a convenient and effective method of monitoring the larynx. A Postcricoid Laryngeal Surface Electrode as herein described shows excellent initial results as an electrode for electromyographic monitoring of the larynx for recurrent laryngeal nerve identification.


Auditory brainstem evoked response in patients undergoing carotid endarterectomy.

Auditory brainstem evoked response-(ABR) may be affected by changes in cerebral blood flow. Apart from its primary indications for prevention of stroke and transient ischemic attacks-(TIA's), internal carotid endarterectomy-(ICE) has been shown to improve cognitive function and cause diminution of tinnitus and vertigo. A one-week postoperative ABR previous study of patients undergoing ICE demonstrated no change in ABR recordings. In order to evaluate possible late changes, we have conducted a prospective study of the delayed effects of ICE on ABR in 13 patients. No initial pathology was noted and no significant change found in any of the absolute and interpeak latencies of ABR waves.


Extrusion rate of Shah and Shepard ventilation tubes in children.

Shah and Shepard ventilation tubes are the two most commonly used ventilation tube in current practice. In some centres these two tubes are used interchangeably, in others the Shepard is often used as the first 'grommet' of insertion and the Shah for subsequent insertions. A study was undertaken of extrusion rate of these ventilation tubes in 180 children who had surgery for Serous otitis media in 1987. Extrusion time was found to be very significantly different between these tubes. The Shah ventilation tube remains in situ 3 months longer than the Shepard. A case is made for the use of Shah ventilation tubes as the first "grommet" of insertion. Otorrhoea following insertion of ventilation tube did not affect the extrusion rate of ventilation tubes in this study. Nor has otorrhoea a predilection for any particular ventilation tube.


A single-fluor approach to DNA sequence determination using high performance capillary electrophoresis.

The Tabor and Richardson strategy for enzymatic chain termination sequencing of DNA using relative peak intensity has been adapted to high performance capillary gel electrophoresis with laser induced fluorescence detection. This approach to DNA sequencing involves the use of only a single fluor and results in significant reduction in the time required to determine a DNA sequence without the use of highly complicated and expensive instrumentation. We present a modification of the Tabor and Richardson approach employing two reactions, each containing complementary mixtures of only three ddNTP's in the concentration ratio 4:2:1. The DNA sequence is determined by relative peak height and by assigning the missing ddNTP to "gaps" between the peaks. The use of only three terminators/reaction simplifies the software task of differentiating between the termination types and makes more efficient use of the available dynamic range. Both complementary mixes generate complete sequence information and the two data files are combined in order to make a more confident sequence call. This process helps to eliminate errors caused by occasional non-uniform incorporation of ddNTP's or false terminations and also alleviates some of the difficulty associated with reading through compressed regions of the electropherogram.


Stability of capillary gels for automated sequencing of DNA.

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)


Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis.

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.


Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus.

A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.


High resolution-separation of DNA bands by electrophoresis with a long gel in a fluorescence-detection DNA sequencer.

A high-resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single-band differentiation of bands of 1040 base DNA.


Compensation for mobility inequalities between lanes computed from band signals in on-line fluorescence DNA sequencing.

Among on-line fluorescence DNA sequencing systems, the four-lane method exhibits the potential for reporting an erroneous sequence due to nonuniform mobility of the DNA fragments migrating among the four lanes. This error is manifest in phenomenon commonly called smiling. This paper presents a computational algorithm which compensates for the mobility inequalities between lanes using signal data obtained from the shorter DNA fragments forming the faster migrating bands. The program mainly consists of two routines: (i) calculation of calibration coefficients (mobility ratios between lanes), and (ii) examination of the coefficients by applying them to a later domain of the same signals. Both routines are connected with several feed-back branches for recalculation. Homology analysis of final sequences has shown that the accuracy rate is maximized with this algorithm and any ambiguous result can be assigned to the residual error inherent in the band identification method used.


cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer.

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.


Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Electrophoresis and detection of tin-labeled DNAs on open-faced gels.

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.


Pulsed field sequencing gel electrophoresis.

The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.


Movement of fluorescence pattern after photobleaching: an accelerated procedure for DNA electrophoretic mobility analysis.

A new approach which is compatible with many of the existing procedures for the analysis of DNA species in gel electrophoresis is being demonstrated. It takes advantage of fluorescence photobleaching in order to create a sharp boundary between the stained and the (partially) photobleached DNA. By arbitrarily creating a stained DNA band of narrower width, the sensitivity to detect (averaged) DNA band movements has been increased. This feature permits measurements of time-dependent electrophoretic mobility over very short time periods. The approach can be used to shorten the running time of gel electrophoresis experiment and to increase the resolution because of the sharper boundary and narrower band width. With faster running time, diffusion of both DNA and dye in the gel also becomes less serious. Movement of fluorescence pattern after photobleaching also permits measurements of localized motions when the gel pores are small in comparison with DNA sizes. Experiments demonstrating some aspects of the proposed technique, as well as the anticipated limitations, are presented and discussed.


Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer.

Although automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10(-17) mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence-collecting yield. The emission and photodestruction cross-sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross-sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence-collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo-destructive, the cross-section of the destruction being about 3.8 x 10(-20) cm2 in buffer solution while that of Texas Red is 1.5 x 10(-21) cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 mW, with an Ar laser (488 nm) for FITC-DNA, and 18 mW, with an He-Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence-collecting efficiency by a factor of 4, and an He-Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra-high sensitivity of 5 x 10(-20) mole/band (S/N-4) was achieved under optimized conditions.


Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.


Fluorescence chemistries for automated primer-directed DNA sequencing.

A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.


High-sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations.

We describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10-50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single-base profiles of four different samples, instead of for the four-base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)-amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional "T" peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.


Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes.

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.


Why can we not sequence thousands of DNA bases on a polyacrylamide gel?

Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field-induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high-field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.


Quantitative single photon emission tomography: verification for sources in an elliptical water phantom.

Accurate absorbed dose calculations are important for a proper dose planning in internal radionuclide therapy. The activity distribution must be measured and the target volume defined. This can be done with single photon emission tomography (SPET) if proper attenuation and scatter correction are employed. This study investigated the calculation of the activity and the volume of different spherical sources. These two parameters are essential for a proper dose calculation. The scatter and attenuation correction method is based on spatially variant scatter functions and density maps. The volume calculation method is based on obtaining a threshold from a grey-level histogram. Both point sources and spheres of different diameters containing technetium-99m were placed in different locations in an elliptical water phantom and imaged by SPET. The activity and the volume of the spheres were calculated from the SPET images and compared with known activities. Results show a quantification of activity within 10% for most of the sources. Important influences on the quantification are (a) the presence of artefacts due to improper reconstruction and (b) the finite spatial resolution which affects the total number of counts within the determined volume.


Role of bone scanning in the management of non-united fractures: a clinical study.

Technetium-99m methylene diphosphonate (99mTc-MDP) bone scintigraphy was performed in 45 patients (42 male and 3 female) with established non-united fractures to predict the healing response to pulsing electromagnetic field stimulation therapy. The bone scans revealed 3 different scintigraphic patterns. The most frequent pattern was an increased uniform uptake of the tracer at the non-union site (group 1). The second pattern was increased activity at the bone ends with a photon-deficient area between the fracture sites (group 2a) or a generalized decrease in the radionuclide concentration in the region of bone fragments (group 2b). When the scintigraphic pattern did not fit either of the two patterns or when the presence of the cold area between the bone fragments could not be judged with certainty, it was called indeterminate (group 3). All patients underwent pulsing electromagnetic field stimulation. The healing rate was 87.5% and 42.8% in group 1 and group 3 patients, respectively. None of the group 2 patients had any evidence of healing, and they all underwent surgical exploration, revealing complicated non-unions. We conclude that 99mTc bone scintigraphy is a useful tool in determining complicated non-unions and selecting the proper therapy mode.


The reliability of the 3-phase bone scan in suspected scaphoid fracture: an inter- and intraobserver variability analysis.

In the diagnosis of scaphoid fracture, the dynamic phase of the radionuclide bone scan alone has been recommended as an early test. To evaluate the independent reliability of the dynamic and static phases of the 3-phase bone scan in this diagnosis, 3 examiners reviewed the 3-phase bone scans of a series of 60 patients with clinical signs of fracture of the carpal scaphoid and with negative or non-diagnostic initial radiographs. The interpretation was performed independently and without the benefit of additional data. The bone scans were reviewed after 1 year by the same observers. The results were analyzed using kappa statistics. The bone scan was suspicious of fracture of the scaphoid in 15 patients. Irrespective of training and experience, the kappa values of the dynamic bone scan between any 2 observers did not exceed 0.57. The kappa values increased significantly when the static phase of the bone scan was examined (> 0.81). The intraobserver variability showed a similar pattern. We conclude that in suspected scaphoid fracture, the dynamic phase of the radionuclide bone scan alone cannot be used as a reliable diagnostic approach because of the low inter- and intraobserver agreement in the interpretation, irrespective of the experience and training of the observer.


Scintigraphic evaluation of disease activity in rheumatoid arthritis: a comparison of technetium-99m human non-specific immunoglobulins, leucocytes and albumin nanocolloids.

Technetium-99m-labelled, non-specific, polyclonal, human immunoglobulin G (99mTc-hIG) has been used to quantify synovial inflammation in rheumatoid arthritis. A comparison was carried out between the scintigraphic results obtained with this tracer, 99mTc-hexamethylpropylene amine oxime-labelled white blood cells (99mTc-WBC) and 99mTc-albumin nanocolloids (99mTc-NC). Twenty patients affected by rheumatoid arthritis and suffering from clinically active synovitis were studied with 99mTc-hIG. The number and sites of the involved joints had been previously assessed on the basis of the presence of pain and/or swelling. A radiological examination had already been carried out on all the joints. Two days after the 99mTc-hIG scan, 10 patients (group 1) underwent 99mTc-WBC scintigraphy and the other 10 (group 2) underwent a 99mTc-NC scan. The results show that the results of 99mTc-hIG and 99mTc-NC scans are in agreement with clinical examinations in the majority of cases. However, a certain number of positive joint scans corresponding to negative clinical examinations was found. The numerical distribution of these results according to the radiological stages seems to show that 99mTc-hIG is more useful than 99mTc-NC in the initial phases of the disease. The 99mTc-WBC scan was negative in a consistent percentage of the joints previously assessed as clinically and 99mTc-hIG scan positive.


Photopenic defects in marrow-containing skeleton on indium-111 leucocyte scintigraphy: prevalence at sites suspected of osteomyelitis and as an incidental finding.

The skeletal distribution of red marrow-containing sites with a decreased uptake of indium-111-labelled leucocytes was examined as part of a retrospective review of 128 consecutive scans in 113 patients. The prevalence of photopenic defects was determined for sites of suspected osteomyelitis and for other skeletal locations included as part of limited or total-body surveys. Of 52 sites suspected of osteomyelitis based upon radiological and clinical data, 21 (40%) demonstrated a decreased leucocyte uptake. The prevalence of photopenia ranged from 79% (11/14) in the spine and 63% (5/8) in the pelvis to 25% (4/16) in the proximal femur and 0% elsewhere in the extremities (0/9) and in the skull (0/3). Fourteen of these 21 defects (67%) were due to active (n = 9) or healed (n = 5) osteomyelitis/discitis. All sites of active osteomyelitis showed destructive changes on correlative radiographs and were associated with infectious processes of more than 1 months duration. Thirty-seven photopenic defects were observed as incidental findings. The prevalence of photopenia as an incidental finding ranged from 0% in the skull, neck and chest to 3%-4% in the thoracolumbar spine and pelvis and 14% in the femoral heads, the latter reflecting primarily bilateral loss of femoral head marrow. No incidentally found photopenic defect reflected active osteomyelitis. At sites in the spine and pelvis with radiologic evidence of bone destruction suggestive of osteomyelitis, an absence of normal red marrow uptake of labelled leucocytes often reflects a variant presentation for active chronic infection. In contrast, incidental photopenia is uncommon at all skeletal sites except the femoral heads, and should not raise concern over unsuspected active osteomyelitis.


Liver transplant rejection and cholestasis: comparison of technetium 99m-diisopropyl iminodiacetic acid hepatobiliary imaging with liver biopsy.

To determine whether the scintigraphic evaluation of technetium-99m diisopropyl iminodiacetic acid (DISIDA) uptake and excretion can distinguish among liver transplant patients with biopsy evidence for rejection, cholestasis or neither condition, we reviewed scintigrams and biopsies in 36 patients. There were 76 scintigrams with corresponding biopsies. Uptake and excretion were graded from image data on scales reflecting normal through severely abnormal values. Biopsies were evaluated for findings of cholestasis and rejection. The majority of scintigrams demonstrated normal uptake (60/75, 80%) and delayed excretion (65/76, 85%), which was most marked immediately after transplantation. One-way analysis of variance showed that the mean excretion values significantly differed between patients with normal biopsies and those with cholestasis and/or rejection (P = 0.0003). However, mean uptake scores demonstrated no statistically significant difference between these two groups of patients (P = 0.1). These findings suggest that 99mTc-DISIDA scintigraphy can differentiate between transplants with and without rejection/cholestasis but not between rejection and cholestasis. If 99mTc-DISIDA excretion is normal, rejection and cholestasis are unlikely.


Myocardial perfusion damage after mediastinal irradiation for Hodgkin's disease: a thallium-201 single photon emission tomography study.

Conflicting data have been reported on the incidence of myocardial abnormalities after mediastinal irradiation for Hodgkin's disease. We studied myocardial perfusion in 31 clinically asymptomatic patients (13 male, 18 female, mean age 35 years) 7 years (range 3-11 years) after mantle field radiotherapy. Thallium-201 tomoscintigraphic data were obtained after exercise, 4 h later and at rest (8-15 days later). Images were analysed visually and quantitatively (sectorial quantification of 201Tl uptake on the bull's eye images of the short-axis slices) compared with those of 35 subjects with a low likelihood of coronary artery disease. Twenty-five tomographic data sets were available. Images were visually abnormal in 21 patients (84%) showing an heterogeneous 201Tl uptake. In 68%, the sectorial 201Tl uptake was lower than the mean 201Tl uptake value minus 2 standard deviations measured in subjects with a low likelihood of coronary artery disease. Significant redistribution (quantitatively assessed > or = 10%) was present in 10 patients (40%). In most of the patients, the location and the shape of the defect(s) could not be anatomically related to an epicardial coronary vessel disease. These results indicate that after mediastinal irradiation the 201Tl myocardial uptake is frequently abnormal. The observed patterns suggest a disease of the small coronary vessels and/or the existence of a myocardial fibrosis rather than epicardial coronary artery disease.


Presurgical identification of hibernating myocardium by combined use of technetium-99m hexakis 2-methoxyisobutylisonitrile single photon emission tomography and fluorine-18 fluoro-2-deoxy-D-glucose positron emission tomography in patients with coronary artery disease.

We tested the possibility of identifying areas of hibernating myocardium by the combined assessment of perfusion and metabolism using single photon emission tomography (SPET) with technetium-99m hexakis 2-methoxyisobutylisonitrile (99mTc-MIBI) and positron emission tomography (PET) with fluorine-18 fluoro-2-deoxy-D-glucose (18F-FDG). Segmental wall motion, perfusion and 18F-FDG uptake were scored in 5 segments in 14 patients with coronary artery disease (CAD), for a total number of 70 segments. Each subject underwent the following studies prior to and following coronary artery bypass grafting (CABG): first-pass radionuclide angiography, electrocardiography gated planar perfusion scintigraphy and SPET perfusion scintigraphy with 99mTc-MIBI and, after 16 h fasting, 18F-FDG/PET metabolic scintigraphy. Wall motion impairment was either decreased or completely reversed by CABG in 95% of the asynergic segments which exhibited 18F-FDG uptake, whereas it was unmodified in 80% of the asynergic segments with no 18F-FDG uptake. A stepwise multiple logistic analysis was carried out on the asynergic segments to estimate the postoperative probability of wall motion improvement on the basis of the preoperative regional perfusion and metabolic scores. The segments with the highest probability (96%) of functional recovery from preoperative asynergy after revascularization were those with a marked 18F-FDG uptake prior to CABG. High probabilities of functional recovery were also estimated for the segments presenting with moderate and low 18F-FDG uptake (92% and 79%, respectively). A low probability of functional recovery (13%) was estimated in the segments with no 18F-FDG uptake.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of myocardial viability with technetium-99m hexakis-2-methoxyisobutyl isonitrile and iodine-123 phenylpentadecanoic acid and single photon emission tomography.

The detection of viable myocardium in infarcted regions, i.e. hibernating myocardium, is a major goal in clinical cardiology today. We applied combined planar and single photon emission tomography (SPET) to the non-invasive estimation of the left ventricular ejection fraction (LVEF), myocardial blood flow and free fatty acid uptake in the heart. Of the 31 patients with coronary artery disease, 25 (81%) had had a previous myocardial infarction. All patients had at least one persistent perfusion defect on the stress-rest technetium-99m hexakis-2-methoxyisobutyl isonitrile (Sestamibi) study, and the results revealed 57/124 (46%) persistent perfusion defects. As a part of the perfusion study, the LVEF was measured at rest using the first-pass 99mTc-Sestamibi injection, and the mean LVEF was 47% +/- 9% (mean +/- 1 standard deviation). Iodine-123 phenylpentadecanoic acid (123I-pPPA) imaging at rest was performed within 2 weeks from the perfusion study. Then 6-mm transaxial, sagittal and coronal slices of the perfusion and 123I-pPPA studies were reconstructed. The bull's eye displays of the coronal slices were visually surveyed and divided into 4 quadrants: anterior, lateral, posterior and septal. The following image score was used: 0 = fixed defect, 1 = partial uptake and 2 = normal uptake. Moreover an index of metabolic reserve (MR) was calculated by dividing the bull's eye of the 123I-pPPA study by the bull's eye of resting 99mTc-Sestamibi, and its maximum value was normalized to 100%.(ABSTRACT TRUNCATED AT 250 WORDS)


Investigation of joint disease.

The role of nuclear medicine in the diagnosis and management of the major arthropathies is critically reviewed, with particular reference to osteoarthritis, rheumatoid and similar forms of arthritis, ankylosing spondylitis, non-specific back pain, gout, the neuropathic joint, avascular necrosis, infection and the consequences of prosthetic joint insertion. Attention is drawn both to practical applications and deficiencies in current techniques and knowledge.


The small intestine and colon: scintigraphic quantitation of motility in health and disease.

Radioisotopes allow accurate quantitation of the pattern and effectiveness of the transit of chyme through the small and large intestines. Abnormalities of small bowel transit can be demonstrated in patients with the irritable bowel syndrome, and patients with chronic idiopathic intestinal pseudo-obstruction due to either a visceral myopathy or neuropathy. In the colon, radioisotopic studies of transit have demonstrated the site of delayed transit in some severely constipated patients. In patients with these disorders of transit, functional studies may influence the choice of medical or surgical therapy although there are few prospective studies which have established their worth in this context. Radioisotope studies can also be utilised to study the effectiveness of delivery of drugs to the small and large bowel, and to study the adequacy of rectal evacuation in patients with a defaecatory disturbance. The low radiation dose and possibility of frequent observations make radioisotope studies valuable for clinical and research studies in functional gastrointestinal disorders.


Merkel cell carcinoma and iodine-131 metaiodobenzylguanidine scan.

Two cases of Merkel cell carcinoma, a neuroendocrine neoplasia of the skin, investigated with iodine-131 metaiodobenzylguanidine (131I-mIBG) scintigraphy, are reported. Uptake in the tumor was evident only in 1 case. The possible diagnostic and therapeutic role of 131I-mIBG in patients with this rare neoplasm is discussed.


Effects of prior use of non-steroidal anti-inflammatory drugs on renal function and transfusion requirements after upper gastrointestinal haemorrhage.

The possibility has been investigated that, after admission to hospital with acute upper gastrointestinal bleeding, patients who have been users of aspirin and non-aspirin non-steroidal anti-inflammatory drugs have poorer baseline renal function, a greater improvement in renal function during their hospital stay, and a larger transfusion requirement than non-users. Patients over 50 years of age admitted to public hospitals with acute upper gastrointestinal bleeding were studied. Creatinine clearance was estimated from serum creatinine and the transfusion requirement was recorded as the number of units of blood transfused on Day 1 and throughout the entire hospital stay. Data were obtained prospectively from case notes and by structured interview. Users of non-steroidal anti-inflammatory drugs were significantly older than non-users. The estimated creatinine clearance on admission to hospital declined with age. Creatinine clearance was 13.2 (95% CI 6.0 to 20.4) ml.min-1 lower in users than non-users of non-aspirin non-steroidal anti-inflammatory drugs. However, the difference was attributable to the older age of the drug users rather than to the drugs themselves. On average, the increase in creatinine clearance during hospital stay was the same in users and non-users of non-aspirin non-steroidal anti-inflammatory drugs. Prior use of aspirin had no effect on any measure of renal function. The incidence of blood transfusion was higher in older than in younger patients but neither the incidence of transfusion, nor the transfusion requirement, was different between users and non-users of non-aspirin non-steroidal anti-inflammatory drugs and aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)


Analgesic efficacy of an ibuprofen-codeine combination in patients with pain after removal of lower third molars.

A double-blind, randomised analgesic trial was carried out in 165 patients undergoing surgical removal of one impacted lower wisdom tooth. In a two-dose regimen, the analgesic efficacy of the combination ibuprofen-codeine 200 mg : 30 mg was compared with that of acetylsalicylic acid-codeine 500 mg : 30 mg and codeine 30 mg. Each dose was taken when the patient needed pain relief. The intensity of the pain was measured on a visual analogue scale during the 10-h period after the first dose. The mean pain reduction by Dose 1 in patients on ibuprofen-codeine, acetylsalicylic acid-codeine and codeine was 64%, 45% and 26%, respectively, and the mean duration of effect was 8.3, 6.3 and 5.6 h. According to the pain reduction, duration of effect and pain reduction index after Doses 1 and 1 + 2, there was a significant difference between ibuprofen-codeine and the other two drugs. The maximum pain reduction within 4 hours was 84% with ibuprofen-codeine. This was significantly different from the reduction achieved both with acetylsalicylic acid-codeine (64%) and codeine (35%). Seventeen patients reported adverse events: 5 on ibuprofen-codeine, 4 on acetylsalicylic acid-codeine and 8 on codeine. The most common events were tiredness and vertigo. It is concluded that the combination ibuprofen-codeine 200 mg : 30 mg had greater analgesic efficacy compared to the combination acetylsalicylic acid-codeine 500 mg : 30 or codeine 30 mg in patients with pain after removal of the lower third molars.


Bromocriptine lessens the incidence of mortality in L-dopa-treated parkinsonian patients: prado-study discontinued.

L-Dopa supplemented by a peripheral decarboxylase inhibitor is considered the most potent therapeutic regimen prolonging active life in Parkinsonian patients. The long-term benefit of therapy is limited by adverse effects, such as dyskinesia and on-off phenomena, which can be mitigated by the concomitant administration of dopamine agonists, such as bromocriptine. In order to quantify the beneficial impact of early combination therapy, a controlled clinical trial (PRADO: PRA videl1 + DO pa) in patients with early Parkinson's disease was carried out, whereby L-Dopa monotherapy (in a fixed combination with benserazide (DoBe) was being compared with the same combination plus bromocriptine (DoBeBro). Patients were recruited and treated by 101 practising neurologists in the Federal Republic of Germany and in Hungary. Twenty seven clinical university centers cross-checked the patients at regular intervals. The trial started with 3 months of DoBe monotherapy (median dose of 375 mg L-Dopa for both randomized groups) followed by gradual substitution of DoBe by bromocriptine over 3 months in one of the groups (250 mg L-Dopa/10 mg bromocriptine). The target medication was maintained from study months 6 to 54. Parkinsonian symptoms were classified according to the Webster rating scale, the Hoehn and Yahr scale and the Zung Self-Rating Depression Scale. Adverse events and life status were checked at regular intervals. Special emphasis was given to motor performance tests. 587 patients (302 in the DoBe group and 285 in the DoBeBro group) were available for intention-to-treat analysis. Both groups were homogeneous at baseline in all observed parameters.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of doxazosin on insulin sensitivity in hypertensive non-insulin dependent diabetic patients.

The effect of doxazosin, an alpha 1-adrenoceptor blocking drug, on blood pressure, sensitivity to insulin and serum lipids has been evaluated in 14 hypertensive, non-insulin dependent diabetic patients. The dose was titrated individually upwards from 1 mg until the diastolic blood pressure was below 90 mm Hg, side-effects precluded further dosage increase or the maximum daily dose of 16 mg was achieved. After 12 weeks of treatment (mean doxazosin dose 5.6 +/- 5.1 mg daily), the supine and standing diastolic blood pressure of the patients had declined by about 7 mm Hg, whereas their systolic blood pressure and heart rate were not significantly changed. The metabolic clearance rate of glucose increased from 2.35 to 3.37 ml.min-1.kg-1 during treatment, suggesting improved sensitivity to insulin. Fasting plasma glucose was 11.9 mmol.l-1 before and 10.9 mmol.l-1 after doxazosin therapy (NS). Serum electrolytes and lipids did not change significantly but serum uric acid decreased from 305 to 281 mumol.l-1. Doxazosin may be a useful alternative for the treatment of hypertension in NIDDM patients.


Changes in Lp(a) lipoprotein levels during the treatment of hypercholesterolaemia with simvastatin.

Thirty-six patients with total serum cholesterol levels above 6.5 mmol/l and Lipoprotein(a) levels above 100 mg.l-1 were evaluated in a 24 week double-blind, placebo controlled, cross-over study to assess the possible changes in Lp(a) during treatment with the HMG CoA reductase inhibitor simvastatin. The median plasma Lp(a) increased from 359 to 464 mg.l-1 during simvastin treatment as compared to placebo (not significant). Individual changes in Lp(a) varied. In a multivariate linear regression analysis the individual change in Lp(a) was correlated with the baseline Lp(a) (r = 0.64), the change in serum triglycerides (r = 0.48) and the baseline apolipoprotein B (r = 0.36). Differences between the Lp(a) phenotypes may explain some of the varied Lp(a) responses. It appears that the effect of simvastatin on the Lp(a) level in individuals is usually insignificant, but in patients with a high Lp(a) simvastatin may further increase it.


General well-being during treatment with different ACE-inhibitors: two double-blind placebo-controlled cross-over studies in healthy volunteers.

Two randomised, double-blind, cross-over studies in healthy volunteers given captopril 50 mg b.d. (n = 37; Study I) or enalapril 20 mg o.d. (n = 40; Study 2) and placebo for 2 weeks have been done to examine general well-being. Subjective experiences were evaluated using the standardised, Minor Symptoms Evaluation-profile (MSEP), which was completed during Run-in and on Days 1, 4, 7 and 14 in the morning. In comparison to placebo and the Run-in period, neither captopril nor enalapril affected the MSEP dimensions of Vitality, Contentment and Sleep. Captopril treatment was also assessed by applying the Quality of Life Clinical Questionnaire during Run-in and on Days 7 and 14. No improvement in the quality of life was demonstrated during treatment in comparison with the placebo or the Run-in period. Thus, no mood elevating effect of the ACE-inhibitors captopril and enalapril was demonstrated in healthy volunteers. Cough, which is believed to be a common adverse effect of ACE-inhibitors, was no more frequent during the treatment with captopril or enalapril than with placebo. It is concluded, that short-term treatment with captopril or enalapril is not perceived differently by healthy volunteers than placebo or no treatment at all. Furthermore, the cough associated with ACE-inhibition may be dependent on the duration of treatment, and two weeks was apparently too short for it to emerge.


The disposition of valproate and its metabolites in the late first trimester and early second trimester of pregnancy in maternal serum, urine, and amniotic fluid: effect of dose, co-medication, and the presence of spina bifida.

We have studied 52 pregnancies in epileptic women taking long-term valproate and have measured the concentrations of the parent compound and 13 of its metabolites by gas chromatography-mass spectrometry in amniotic fluid, maternal serum, and 24 h maternal urine samples. All metabolites of valproate present in the serum could also be detected in the amniotic fluid, although at much lower concentrations. Amniotic fluid concentrations of valproate and several of its metabolites ((E) delta 2-valproate, (2E,3'E) delta 2,3'-valproate, and 3-keto-valproate) correlated with total valproate concentrations as well as with unbound valproate concentrations in maternal serum. We suggest that the amniotic fluid acts as a deep compartment, with slow appearance and disappearance of valproate and its main metabolites. The data further suggest that during the first and early second trimesters of pregnancy the beta-oxidation of valproate decreases. In pregnancies associated with fetal neural tube defects (n = 5) significantly higher daily doses of valproate were used compared with normal pregnancies (n = 47). This resulted in higher concentrations of valproate in maternal serum. However, the metabolite patterns in maternal serum, 24 h urine samples, and amniotic fluid did not show any significant differences in pregnancies with neural tube defects.


Phenytoin metabolism during pregnancy.

The steady-state 72 h urinary excretion of various phenytoin metabolites has been measured in 10 epileptic women, whose plasma phenytoin concentrations relative to the phenytoin dose fell during pregnancy and rose again post-partum. In later pregnancy and post partum, a mean of 61.3% and 48.9%, respectively, of the total daily phenytoin dose was eliminated as 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). Even though p-HPPH accounts for not much more than half the total daily phenytoin dose, increased excretion of this metabolite sufficed to account for the elimination of the entire increase in the dose of phenytoin required during pregnancy. There was no definite increase in the excretion of any other (minor) metabolite measured. Thus pregnancy seems not to enhance uniformly the capacity of the various metabolic pathways of phenytoin.


Carbamazepine kinetics and adverse effects during and after ethanol exposure in alcoholics and in healthy volunteers.

The influence of ethanol on the single-dose kinetics of carbamazepine (400 mg syrup) was assessed in 7 alcoholics after a debauche (mean daily consumption 240 g ethanol) and after 9 days of controlled abstinence, and in 8 healthy volunteers after intake of the drug with and without a single dose of ethanol (25 g). Twelve h after the first test dose of carbamazepine the alcoholics were treated with the drug for 4 days (200 mg tablet b.d.). Carbamazepine was then withheld until a single test dose was given on day 9. Serum levels of carbamazepine and its 10,11-epoxide metabolite were measured by liquid chromatography. Carbamazepine absorption appeared to be delayed in alcoholics, both after debauche and withdrawal, but its bioavailability did not seem to be reduced. Carbamazepine levels were higher and those of its metabolite lower in alcoholics after a debauche than after 9 days of controlled abstinence, but neither was changed in healthy volunteers after the ingestion of carbamazepine together with a single dose of ethanol. The difference may have been due to inhibition of carbamazepine metabolism by ethanol at the high levels attained in alcoholics but not in volunteers. However, it could also be an expression of the unmasking of enzyme induction after ethanol withdrawal. None of the alcoholics had any withdrawal seizures. Despite similar carbamazepine levels, side effects occurred in all volunteers but in none of the alcoholics, indicating that long-term ethanol exposure may promote central nervous adaptation to the acute untoward effects of carbamazepine.(ABSTRACT TRUNCATED AT 250 WORDS)


Prediction of phenotype for acetylation and for debrisoquine hydroxylation by DNA-tests in healthy human volunteers.

The debrisoquine/sparteine-type polymorphism of drug oxidation and the polymorphism for acetylation are two common inherited variations in human drug metabolism. The phenotypes for hydroxylation and acetylation can be predicted be newly developed methods based on mutation-specific amplification of DNA by the polymerase chain reaction (PCR), which also allow for identification of heterozygous carriers of one mutant allele. In the present study, the results of genotyping of 81 healthy European volunteers were compared with the phenotype obtained by the classical biochemical approach using debrisoquine and caffeine as probe drugs. Genotyping correctly predicted all 73 extensive metabolisers (EMs) and 6 out of 8 poor metabolisers (PMs) of debrisoquine. All 48 rapid acetylators and 33 of 35 slow acetylators were predicted. Overall, the DNA analysis result matched the in vivo phenotype in 97.5% of individuals.


Steady-state plasma levels of clomipramine and its metabolites: impact of the sparteine/debrisoquine oxidation polymorphism. Danish University Antidepressant Group.

After an initial placebo week, 37 depressed inpatients were treated with the fixed dose of 75 mg clomipramine b.d. A sparteine test was carried out during the placebo period and again during the second week of active therapy. Blood for drug assay was collected at the end of the inter-dose interval in the (morning) at weekly intervals. Clomipramine and four metabolites (desmethylclomipramine, didesmethylclomipramine, 8-hydroxyclomipramine, and 8-hydroxydesmethylclomipramine) in plasma were assayed by reversed phase HPLC. The clomipramine and desmethylclomipramine steady-state plasma levels varied by factors of 11 and 9, respectively, and the clomipramine/8-hydroxyclomipramine and desmethylclomipramine/8-hydroxydesmethylclomipramine ratios both varied by 7-fold. During the placebo week, 36 patients were phenotyped as extensive metabolizers (EM) (metabolic ratio, MR, 0.1-2.0), and one patient was phenotyped as a poor metabolizer (PM) (MR > 300). During clomipramine treatment, one patient changed phenotype from EM to PM (MR = 140). In the EM, the median of the MR increased from 0.4 to 2.3. There was a statistically significant correlation between the MR before and during clomipramine treatment, even when the PM was excluded. Neither the steady-state plasma clomipramine levels nor the clomipramine/desmethylclomipramine ratios showed a significant correlation with the MR. In contrast, the desmethylclomipramine and didesmethylclomipramine steady-state levels and the desmethylclomipramine/8-hydroxydesmethylclomipramine and clomipramine/8-hydroxyclomipramine ratios showed a significant positive correlation with the MR. The PM had the highest steady-state plasma desmethylclomipramine level and the highest desmethylclomipramine/8-hydroxydesmethylclomipramine ratio. These correlation coefficients (rs) were generally increased when the correlation analyses were based on the MR obtained during clomipramine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Drug utilization at primary health care level in southern India.

Primary health centres provide health care to the majority of the population in developing countries. A drug utilization study was conducted for 1 y at two primary health centres in Pondicherry, India. Information on complaints, diagnosis and drugs prescribed was collected. From the 2953 prescriptions studied, it was found that on an average each patient received 2.71 drugs. Vitamins, antibiotics, analgesics and antihistamines were the most commonly used, accounting for more than 80% of the drugs prescribed. The antimicrobials which constituted one fourth of the drug consumption, comprised sulphonamides, tetracycline and cotrimoxazole. About half of the patients received injections, particularly of the vitamin B Complex and antibiotics. The results will be used to plan intervention strategies for the promotion of rational drug use.


Pharmacokinetics of carteolol in patients with impaired renal function.

In order to determine the appropriate dosage of carteolol in renal dysfunction, the pharmacokinetics of carteolol has been examined in appropriate patients. The plasma concentrations and urinary excretion of carteolol were investigated in 15 patients with varying degrees of renal impairment during the administration of 5-20 mg carteolol hydrochloride (5 mg/tablet) for 2-45 months. Plasma carteolol levels were linearly correlated with the serum creatinine concentration (r = 0.87) and reciprocally with the creatinine clearance (r = 0.82). The urinary carteolol concentration was correlated with the urinary creatinine concentration (r = 0.69) and the urinary carteolol excretion was also correlated with the creatinine clearance (r = 0.79). These relationships become even closer when the plasma carteolol concentrations and urinary excretion rate of carteolol were factored by the administered tablets. The fractional renal excretion of carteolol was virtually constant at various degrees of renal function, and it always exceeded 100%, which indicates that carteolol was actively secreted, even in patients with renal failure. The estimated tubular secretion rate of carteolol was logarithmically correlated with the fractional renal excretion of carteolol (r = 0.93). The results indicate that the dose of carteolol should be determined according to the degree of renal impairment.


Uptake and localisation of O-(beta-hydroxyethyl)-rutosides in the venous wall, measured by laser scanning microscopy.

The uptake and localisation of O-(beta-hydroxyethyl)-rutosides (HR) in the venous wall was studied in 8 patients undergoing crossectomy for a varicose long saphenous vein. The fluorescence of cross-sections of the vein wall was measured by laser scanning microscopy, based on the autofluorescence of HR. Four patients (treated group) received 2 x 1.5 g HR IV before surgery, and four (untreated patients) served as controls. Uptake of HR into the veins from treated patients was seen, with a mean fluorescence intensity of 80.9 units compared to 49.4 units in the untreated veins. The increase in fluorescence was clearly demarcated on the endothelial side of the vein wall. It is concluded that HR passes into the vascular wall, where it is localised in the endothelial and sub-endothelial areas.


Plasma drug profiles and tolerability of MK-571 (L-660,711), a leukotriene D4 receptor antagonist, in man.

We have studied the tolerability and plasma drug profiles of a leukotriene D4 receptor antagonist, MK-571, given intravenously and as an oral solution in two separate trials. Study I (i.v.) involved 2 panels of 6 healthy men in a double-blind, alternating, incrementally increasing dose study with single doses up to 1500 mg. There was good tolerability at all doses. Plasma was assayed stereospecifically by HPLC for the S(+) and R(-) enantiomers of MK-571. For each enantiomer AUC values increased more than proportionately with increasing dose, suggesting nonlinear kinetics. The S(+) enantiomer was cleared more rapidly than the R(-) enantiomer. The apparent initial volume of distribution was less than 101 for both enantiomers. Study II (oral) involved 18 healthy subjects in 3 parallel groups who took multiple oral doses of 100, 300, and 600 mg t.i.d. for 31 doses. MK-571 administration was well tolerated, with only mild to moderate gastrointestinal discomfort at the highest dose. Total MK-571 (plasma samples assayed nonstereoselectively) was rapidly absorbed after oral administration, reaching peak concentrations at 1-2 h. Mean 8 h AUC increased from dose 1 to dose 31 in all subjects at all doses, suggesting a modest extent of accumulation (about 50%) of total MK-571 in plasma with a t.i.d. dosage regimen.


Dose-dependent kinetics of the enantiomers of MK-571, and LTD4-receptor antagonist.

The disposition of the enantiomers of MK-571 (MK-0679 and L-668,018) following single i.v. doses of MK-571 (L-660,711) was studied in a three way cross-over study in 12 healthy male volunteers. Each volunteer received 75 mg, 300 mg and 600 mg i.v. doses of MK-571 at weekly intervals. The disposition of both enantiomers appeared dose-dependent, since the AUC increased disproportionately faster than the dose. The dose dependency was much more pronounced for L-668,018: its AUC increased 6-fold from the 75 to the 300 mg dose, 16-fold from 75 to 600 mg and 2.7 fold from 300 to 600 mg. For MK-0679, the corresponding increases in AUC were 4.8-, 11-, and 2.3 fold. Regardless of dose, the elimination of L-668,018 was more rapid than that of MK-0679. The disposition of MK-0679 needs to be investigated independently to detect any potential influence of L-668,018 on its disposition.


Population pharmacokinetic parameters for Bayesian monitoring of amikacin therapy in intensive care unit patients.

The pharmacokinetics of amikacin has been studied in 40 intensive care unit patients using the bayesian estimation method implemented in the USC PC PACK program of Jelliffe. The volume of the central compartment was significantly higher in these patients than in the reference population, while other pharmacokinetic parameters did not differ significantly from the reference values. The population values may be employed, in addition to those supplied with the software, to adapt dosage regimens of amikacin in ICU patients.


Pharmacokinetics of pirmenol in young and elderly subjects.

The steady state pharmacokinetics of pirmenol was compared in twelve healthy young (aged 18 to 45 y) and 11 elderly subjects (over 65 y) subjects given pirmenol HCl 100 mg every 12 h for a total of 14 doses. In addition, the single-dose pharmacokinetics of pirmenol was determined following a 100 mg oral dose in the young subject group for comparison with the results of repeated administration. In the young subjects, the mean single-dose and steady-state CLR of pirmenol were similar; however, Ae was 29% higher and CL/f was 22% lower at steady state than after the single dose. Steady-state (fourteenth dose) Cmin, Cmax, tmax, lambda z, Ae, CL/f, CLR and V values were similar in the young and elderly subjects. Based on pharmacokinetic considerations, the dosage of pirmenol is unlikely to differ in young and elderly subjects.


The effect of a low dose of quinidine on the disposition of flecainide in healthy volunteers.

We have studied the effects of quinidine on ECG intervals and on the pharmacokinetics of flecainide and its two metabolites in 6 healthy men in an open randomized crossover study. Flecainide acetate (150 mg) was given as a constant rate i.v. infusion over 30 min. Quinidine (50 mg orally), given the previous evening, did not change the volume of distribution of flecainide (7.9 vs 7.4 l.kg-1), but significantly increased its half-life (8.8 vs 10.7 h). This was attributable to a reduction in total clearance (10.6 vs 8.1 ml.min-1 x kg-1), most of it being accounted for by a reduction in non-renal clearance (7.2 vs 5.2 ml.min-1 x kg-1). The excretion of the metabolites of flecainide over 48 h was significantly reduced. These findings suggest that quinidine inhibits the first step of flecainide metabolism, although it may also reduce its renal clearance, but to a lesser extent (3.5 vs 2.9 ml.min-1 x kg-1). The effects of flecainide on ECG intervals were not altered by quinidine. Thus, quinidine tends to shift extensive metabolizer status for flecainide towards poor metabolizer status and may also alter its renal excretion.


Captopril does not interact with the pharmacodynamics and pharmacokinetics of digitoxin in healthy man.

The chronic oral administration of 0.07 mg digitoxin o.d. for up to 58 days to 12 healthy volunteers caused a small drop in mean heart rate HR (95% CI: -7.9 to -1.6 beats.min-1), in mean diastolic blood pressure (95% CI: -8.3 to -0.4 mmHg), shortening of the QTc-interval (95% CI: -42 to -19 ms), shortening of the HR-corrected pre-ejection period PEPc (95% CI: -16 to -1 ms) and electromechanical systole QS2c (95% CI: -25 to -1 ms), and an increase in the impedance cardiographic Heather index (dZ/dtmax/RZ, 95% CI: 0.3 to 4.3) relative to the baseline measurements before digitalisation. The concomitant administration of 25 mg oral captopril b.d. did not significantly alter these responses relative to the concomitant double-blind administration of placebo, nor did it alter the pharmacokinetic characteristics of plasma digitoxin at steady state. Thus, no relevant change in the pharmacokinetic and pharmacodynamic characteristics of chronically administered digitoxin were induced by concomitant treatment with captopril.


Effect of N(G)-nitro-L-arginine methyl ester on the hypotensive and hypertensive responses to 5-hydroxytryptamine in pithed rats.

The potential ability of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 1 to 10 mg/kg), to modulate blood pressure responses to 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT) and (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (DOI) was studied in pithed rats. The hypotensive responses to 5-HT in the presence of ketanserin were augmented by L-NAME as well as by phenylephrine infusion, both of which themselves increased blood pressure. L-NAME also tended to prolong the duration of the response of 5-HT. Likewise, the hypotensive responses to 5-CT were potentiated. The magnitude of the hypertensive responses to 5-HT was unaffected by L-NAME or phenylephrine. However, in contrast to phenylephrine, L-NAME prolonged the duration of these responses. The magnitude and duration (middle dose only) of the hypertensive responses to DOI were augmented by L-NAME, but phenylephrine was ineffective. These results suggest that L-NAME increases blood pressure, probably by inhibiting the basal release of NO in animals with a low vascular tone. However, the hypotensive responses to 5-HT and 5-CT seem to be largely independent of NO release by the endothelium, but the hypertensive responses to 5-HT and DOI appear to be limited by the release of NO in the pithed rat.


ucb L059, a novel anti-convulsant drug: pharmacological profile in animals.

The anticonvulsant activity of ucb L059 ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide) was evaluated in a range of animal models. ucb L059 was active after oral and intraperitoneal administration in both rats and mice, with a unique profile of action incorporating features in common with several different types of antiepileptic drugs. The compound was active, with ED50 values generally within the range of 5.0-30.0 mg/kg, in inhibiting audiogenic seizures, electrically induced convulsions and convulsions induced chemically by pentylenetetrazole (PTZ), bicuculline, picrotoxin and N-methyl-D-aspartate (NMDA). ucb L059 retarded the development of PTZ-induced kindling in mice and reduced PTZ-induced EEG spike wave discharge in rats. The R enantiomer, ucb L060, had low intrinsic anticonvulsant activity, showing the stereospecificity of action of the molecule although the actual mechanism of action remains unknown. Neurotoxicity, evaluated with an Irwin-type observation test, the rotarod test and open-field exploration, was minimal, with only mild sedation being observed, even at doses 50-100 times higher than the anticonvulsant doses; at pharmacologically active doses, the animals appeared calm but slightly more active. ucb L059 thus presents as an orally active, safe, broad-spectrum anticonvulsant agent, with potential antiepileptogenic and anti-absence actions.


Effects of phorbol ester on lower urinary tract smooth muscles in rabbits.

The contractile effects of phorbol 12,13-dibutyrate (PDBu) on rabbit urinary bladder dome and urethra were investigated using muscle bath techniques. PDBu caused concentration-dependent contractions in both tissues, and these responses were not affected by pretreatment with atropine, phentolamine, hexamethonium or indomethacin. In both tissues, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), inhibited PDBu-induced contractions in a concentration-dependent manner. The maximum PDBu-induced contractions in bladder dome and urethra were 33.5 +/- 3.4 and 33.3 +/- 4.1% of KCl-induced maximum contractions, respectively. In Ca(2+)-free solution or after pretreatment with nifedipine (10(-6) M), PDBu-induced contractions were reduced but not completely abolished. Although pretreatment with PDBu (10(-8) M) did not have a significant effect on the contractile responses induced by carbachol (in bladder dome) and phenylephrine (in urethra), pretreatment with H-7 (100 microM) had an inhibitory effect on carbachol- and phenylephrine-induced contractions; tonic phase contractions were more sensitive than phasic contractions. These results indicate that PDBu has significant contractile effects in rabbit bladder dome and urethra, and that the effects may be partly mediated by activation of PKC. PKC activation might also contribute to agonist-induced contractile responses in these tissues.


Effect of Mn2+ on neonatal and adult rat heart: initial depression and late augmentation of contractile force.

In the present study, we examined the inotropic effect of Mn2+ on adult and neonatal rat myocardia, contraction of which is known to be highly dependent on Ca2+ release from the sarcoplasmic reticulum and trans-sarcolemmal Ca2+ influx, respectively. Mn2+ produced an initial negative inotropic effect followed by a late augmentation of contractile force in both neonatal and adult preparations, accompanied by marked prolongation of the contraction duration. The attenuation of the late augmentation by ryanodine was greater in the adult while the effect of nicardipine was greater in the neonate, which was similar to the effects of the two drugs in the absence of Mn2+. We tentatively concluded that Mn2+ produces late augmentation of the contractile force in neonatal and adult rat myocardia through some action on the general mechanism of force development, rather than by acting specifically on the sarcoplasmic reticulum.


Dopaminergic modulation of pilocarpine-induced motor seizures in the rat: the role of hippocampal dopamine D1 receptors.

The present study addressed the role of dopamine D1 receptors in pilocarpine-induced motor seizures in rats. Bilateral pretreatment of the hippocampus with the D1 agonist SKF 38393 (0.1-5 micrograms) did not alter the animals' sensitivity to a threshold (200 mg/kg i.p.) or fully convulsant dose (600 mg/kg i.p.) of pilocarpine, as compared to hippocampal saline-treated controls. Similarly, direct injection of pilocarpine (200 micrograms per side) into both hippocampi elicited low level seizure activity that was not modified by SKF 38393, either coadministered (2 micrograms per side) or injected systemically (30 mg/kg i.p.). On the other hand, intrahippocampal microinjections of the D1 antagonist, SCH 23390 (2 micrograms per side), whilst unable to prevent epileptogenesis to 600 mg/kg pilocarpine, delayed the onset of seizures and reduced their severity. These results suggest that hippocampal dopamine lowers the seizure threshold by activating D1 receptors, an effect which is only disclosed by D1 receptor blockade and is not surmountable by additional D1 stimulation.


Influence of pertussis toxin on thermic responses to morphine and neurotensin in rats.

The influence of pertussis toxin (PTX) on thermic responses elicited by morphine and neurotensin was evaluated in unrestrained rats kept at 22 degrees C. High doses of morphine (9-36 micrograms/rat i.c.v.) lowered body temperature and low doses (1.25, 2.5 micrograms/rat i.c.v.) produced hyperthermia. The hyperthermic effect was more resistant than the hypothermic effect to naloxone antagonism. Neurotensin (50, 100 micrograms/rat i.c.v.) induced marked hypothermia followed by hyperthermia. I.c.v. injection of PTX (1 microgram), six days before morphine (18 micrograms/rat i.c.v.), replaced the opiate hypothermia by consistent hyperthermia and reduced by 60% the hyperthermia elicited by morphine (2.5 micrograms/rat i.c.v.). The toxin also affected the thermic responses induced by neurotensin (50 micrograms/rat i.c.v.) administered six days after PTX (1 microgram/rat i.c.v.). The initial hypothermia was enhanced by 173% and the late hyperthermia was fully antagonized. It thus appears that PTX-sensitive G-proteins play different roles in the molecular events underlying the thermoregulatory responses to morphine and neurotensin.


Pancopride, a potent and long-acting 5-HT3 receptor antagonist, is orally effective against anticancer drug-evoked emesis.

Pancopride ((+-)N-(1-azabicyclo-[2,2,2]-oct-3-yl)-2-cyclopropylmethoxy-4-ami no-5-chlorobenzamide) is a new potent and selective 5-HT3 receptor antagonist, orally and parenterally effective against cytotoxic drug-induced emesis. In vitro, pancopride displayed high affinity (Ki = 0.40 nM) for [3H]GR65630-labelled 5-HT3 recognition sites in membranes from the cortex of rat brains. In vivo, pancopride antagonized 5-HT-induced bradycardia in anaesthetized rats when administered i.v. 5 min (ID50 = 0.56 microgram/kg) or p.o. 60 min (ID50 = 8.7 micrograms/kg) before 5-HT challenge. A single oral dose (10 micrograms/kg) of pancopride produced a significant inhibition of the bradycardic reflex over an 8-h period. Pancopride dose dependently inhibited the number of vomiting episodes and delayed the onset of vomiting induced by cisplatin in dogs (ID50 = 3.6 micrograms/kg i.v. and 7.1 micrograms/kg p.o.). Pancopride was also effective in blocking mechlorethamine- and dacarbazine-induced emesis. Unlike metoclopramide, pancopride was shown to lack any measurable antidopaminergic activity both in vitro and in vivo. These results support clinical data, indicating that pancopride will be a useful drug for treating cytostatic-induced emesis in humans.


Dexamethasone attenuates altered insulin secretion elicited by interleukin-1 beta in HIT cells.

The effects of dexamethasone on the modulation of insulin secretion by recombinant human interleukin-1 beta (IL-1) were examined in HIT-T 15 cells. The addition of IL-1 from 1.2 x 10(-8) to 10(-10) M increased insulin secretion in the 0-4-h period after IL-1 administration and prostaglandin E2 (PGE2) production was suppressed by IL-1 from 1.2 x 10(-8) to 10(-12) M. At all doses used, IL-1 inhibited insulin secretion in the 4-24-h period after IL-1 administration and PGE2 levels were increased in the culture medium. In the second experiment, the addition of 10(-7) M dexamethasone prevented the inhibitory effects of IL-1 on insulin secretion. In the third experiment, dexamethasone at 10(-7) M attenuated both the short-term stimulation of insulin release and the long-term suppression of insulin release caused by IL-1. It also prevented the effects of IL-1 on PGE2 production. The present studies suggest that dexamethasone may have a suppressive action on the effects of IL-1 on in vitro insulin secretion.


The vasoconstrictor action of big endothelin-1 is phosphoramidon-sensitive in rabbit saphenous artery, but not in saphenous vein.

The effects of big endothelin-1 and endothelin-1 on vascular reactivity were compared in isolated rabbit saphenous artery and vein. The contractile potency of endothelin-1 was three times higher in the vein than in the artery. In contrast, big endothelin-1 was two times more effective in the artery. Phosphoramidon (100 microM), a metalloproteinase inhibitor, antagonised the contractile action of big endothelin-1 in the artery but not in the vein. These data suggest that the biosynthetic pathway leading to the formation of endothelin differs in arterial and venous vessels.


Effects of baclofen on amino acid release.

This study showed the effects of baclofen on endogenous excitatory (Glu and Asp) and non-excitatory (Tau, Gly and Ala) amino acid release. (A) Release was stimulated by K+ 30 mM in rat frontal cortex slices in vitro (evoked release in ng/g tissue per 5 min): 3739 +/- 215 (Asp), 3429 +/- 357 (Glu), 763 +/- 181 (Tau), 945 +/- 71 (Ala), 468 +/- 44 (Gly). (B) Release was largely Ca(2+)-dependent for all amino acids but Ca(2+)-independent release was observed for Asp and Glu (29 and 32%, of total evoked release respectively). (C) Ca(2+)-dependent release was inhibited by baclofen 100 microM (% of inhibition taking as 100%, the Ca(2+)-dependent release in the absence of baclofen): 46 (Asp), 96 (Glu) (85% inhibition by baclofen 10 microM), 100 (Tau), 77 (Ala). Ca(2+)-independent release was inhibited: 87 and 88% (Asp), 85 and 95% (Glu) by baclofen 10 and 100 microM respectively. It is concluded that baclofen at a high concentration inhibits the evoked release of Glu, Asp, Tau and Ala due to inhibition of the Ca(2+)-dependent fraction and also of the Ca(2+)-independent component in the case of Glu and Asp. Baclofen at a low concentration inhibits only Glu- and Asp-evoked release (total release) due to inhibition of both Ca(2+)-dependent and -independent fractions in the case of Glu and to inhibition of the Ca(2+)-independent fraction in the case of Asp. The possibility that baclofen might affect the Ca(2+)-independent carrier-mediated release of Glu-Asp is discussed.


Involvement of haloperidol-sensitive sigma-sites in antitussive effects.

The effects of selective sigma-ligands on the capsaicin-induced cough reflex in rats were studied. Intraperitoneal injection of (+)-N-allylnormetazocine ((+)-SKF-10,047) and N,N'-di(ortho-tolyl)guanidine (DTG) in doses that ranged from 0.3 to 3.0 mg/kg decreased the number of coughs dose dependently. The antitussive effects of these sigma-ligands were significantly attenuated by pretreatment with haloperidol. Pretreatment with haloperidol also markedly reduced the antitussive effects of (+/-)-pentazocine and dextromethorphan. These results suggest that haloperidol-sensitive sigma-sites may be involved in the regulation of coughs.


Investigation into the 5-hydroxytryptamine-induced relaxation of the circular smooth muscle of guinea-pig stomach fundus.

The 5-HT receptor that mediates relaxation of circular muscle strips of the guinea-pig stomach fundus under resting tone was investigated. Concentration-dependent relaxation was obtained in the presence of atropine (0.2 microM) with 5-hydroxytryptamine (5-HT) (apparent mean pEC50 value, 5.27), 5-carboxamidotryptamine (7.35), 5-methoxytryptamine (4.98) and 5-methyltryptamine (4.58). 1-(m-Trifluoromethyl-phenyl)piperazine and 8-hydroxy-2-(di-n- propylamino)tetralin acted as partial agonists while 2-methyl-5-hydroxytryptamine, alpha-methyl-5-hydroxytryptamine, sumatriptan, metoclopramide and cisapride had little or no effect on the guinea-pig stomach fundus. The concentration-response curve for 5-HT was not affected by tetrodotoxin (0.3 microM), guanethidine (5 microM) or indomethacin (2 microM), suggesting that the relaxation is non-neuronal in origin and is independent of the release of catecholamines or prostanoids. The non-selective 5-HT receptor antagonist, metitepine (0.03-0.1 microM), the 5-HT1C/5-HT2 receptor antagonists, mianserin (0.3-1 microM), pizotifen (0.3-1 microM), ketanserin (3-10 microM), and the 5-HT1A/5-HT2 receptor antagonist, spiperone (3 microM), shifted the concentration-response curves for 5-HT to the right. A 5-HT3 receptor antagonist, ICS205-930 (1 microM), propranolol (1 microM) and phentolamine (1 microM) failed to block the 5-HT-induced relaxation. In conclusion, the results found with agonists and antagonists are compatible with the view that a 5-HT1-like receptor is involved in 5-HT-induced direct relaxation of circular muscle of guinea-pig stomach fundus.


Neuroprotective effect of 5-HT3 receptor antagonist on ischemia-induced decrease in CA1 field potential in rat hippocampal slices.

The effect of 5-HT3 receptor agonists and antagonists on the hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 field potential elicited by stimulation of Schaffer collaterals was investigated using rat hippocampal slices. Treatment with the 5-HT3 receptor agonist, 2-methyl-5-HT (1-10 microM), exacerbated the ischemia-induced decreased in CA1 field potential, whereas treatment with the 5-HT3 receptor antagonist, Y-25130 (0.1-100 microM), or the 5-HT2 receptor antagonist, ketanserin (10, 100 microM), produced dose-dependent neuroprotection against the ischemia-induced decrease. However, in normal non-ischemic solution these treatments did not significantly change the CA1 field potential. The protective action of Y-25130 was blocked by co-treatment with 2-methyl-5-HT. The magnitude of protection in the Y-25130-treated group (EC50, 1.8 microM) was about 20 times greater than that in the ketanserin-treated group (EC50, 33 microM). The present study demonstrated that stimulation of 5-HT3 receptors plays a detrimental role in the development of ischemic damage, whereas blockade of the 5-HT3 receptor plays a neuroprotective role in ischemic damage, suggesting a facilitatory role of 5-HT neurons in ischemia-induced neuronal deficits.


Effects of ouabain on muscle tension and intracellular Ca2+ level in guinea-pig aorta.

The effects of ouabain on muscle tension and the intracellular Ca2+ level ([Ca2+]i) were examined in guinea-pig aorta loaded with fura-2. Ouabain caused a gradual and sustained increase in both [Ca2+]i and muscle tension. There was a positive correlation between these two parameters. In Ca(2+)-free solution, ouabain did not affect either [Ca2+]i or muscle tension, suggesting that the ouabain-induced increase in [Ca2+]i was not due to Ca2+ release from storage sites. The ouabain-induced increase in [Ca2+]i and muscle tension was inhibited by Ni2+, which inhibits the Na+/Ca2+ exchanger, but not by verapamil. Furthermore, anionic and cationic amphiphiles were used as modulators of the Na+/Ca2+ exchanger. Sodium dodecyl sulfate accelerated the responses to ouabain, whereas dodecyltrimethylammonium bromide inhibited them. These results suggest that in the guinea-pig aorta, ouabain induces contraction by increasing the Ca2+ influx through the Na+/Ca2+ exchanger on the plasma membrane, but not through verapamil-sensitive Ca2+ channels.


Analgesic effect of salmon-calcitonin administered by two routes. Effect on morphine analgesia.

We compared the analgesia induced by intraperitoneally (i.p.) and intracerebroventricularly (i.c.v.) administered salmon-calcitonin (S-CT), using the hot-plate test and the writhing test. The influence of the route of administration on the analgesia induced by morphine was also studied. After i.p. administration the analgesic effect was observed only in the writhing test. When S-CT was administered i.c.v., analgesia was observed in both tests, although it was greater in the writhing test than in the hot-plate test. I.c.v. injected S-CT increased the analgesia of i.p. injected morphine. Our results provide new information about the analgesic effect of S-CT and suggest that central mechanisms are involved.


Action of externally applied ATP on rat reticulospinal vasomotor neurons.

In anesthetized rats, iontophoretic application of ATP excited the spinal cord-projection neurons in the rostral ventrolateral reticular nucleus of the medulla oblongata. The neuronal response to ATP was mimicked and then blocked by alpha,beta-methylene-ATP, a metabolically stable ATP analogue, and the response was abolished by suramin. Microinjections of ATP (3-100 pmol) into the rostral ventrolateral medulla produced a powerful pressor response. The results suggest that ATP may function as a neurotransmitter or neuromodulator involved in medullary regulation of cardiovascular functions.


D-cycloserine reverses the working memory impairment of hippocampal-lesioned rats in a spatial learning task.

It is shown that D-cycloserine has cognition-enhancing properties in quinolinic acid hippocampal-lesioned rats. Lesioned rats had a severe impairment of working memory in an allocentric spatial reversal paradigm in the 8-arm maze, and performance could be restored with 12 mg/kg i.p. D-cycloserine, given 30 min before testing. The present findings favour the testing of D-cycloserine for clinical efficacy in patients with Alzheimer's disease with loss of pyramidal neurons and disconnected glycine-NMDA receptor activation.


Ethanol can inhibit nitric oxide production.

Endogenous nitric oxide (NO) in exhaled air from anaesthetized rabbits was monitored by chemiluminescence. Ethanol (3-30 mmol kg-1) infused i.v. dose dependently reduced the levels of exhaled NO, with an IC50 of 23 +/- 3 mmol kg-1. L-Arginine (1 g kg-1) did not reverse the effect of ethanol. These results demonstrate the inhibition of NO formation by ethanol in vivo.


Postnatal behavioral development in methylazoxymethanol-induced microcephalic rats--a behavioral teratology study.

Behavioral testings in methylazoxymethanol (MAM)-induced microcephalic rats were conducted. Pregnant Sprague-Dawley rats were treated intraperitoneally with 0, 20 or 40 mg/kg of MAM once a day on day 14 of gestation and were allowed to delivery. Male pups from each litter were examined for open field test at 6 weeks of age and shuttle-box avoidance test at 7 weeks or more of age. In the open field activity of pups, the counts of ambulation and locomoting distance in 40 mg/kg group have increased significantly as compared with those in control group. In the shuttle-box avoidance test, the avoidance response rate was dose-dependently high in the session of the 1st day. As to the interaction between the avoidance response rate and sequence of sessions, however, the avoidance response rate in 40 mg/kg group was significantly low. Rate of the rats with errors and number of response during the intertrial interval was significantly high in 40 mg/kg group. Thus, we could demonstrate functional disturbance in the memory retaining ability in utero MAM-exposed rats.


[Biological effects of exposure to high frequency electromagnetics on rabbits and guinea pigs].

To investigate the biological effects of exposure to feeble high frequency electromagnetism, skin surface temperature, blood vessel (arterioles and venules) diameter were examined, using infrared thermography, a laser doppler flowmeter, and a video microscope, respectively, in the ear of rabbits. After exposing the ear of rabbits to high frequency electromagnetism value of 9 MHz for 15 minutes, continued rising of local temperature was demonstrated. Though dilatation of arterioles was not seen. In addition, venules tended to dilate and blood flow also to increase, and microcirculation was accelerated at the site where electromagnetism was exposed. Hazardous effects of long term exposures of high frequency electromagnetism (9 MHz for 30 days, 8 hours/day) on guinea pigs were not observed in their behavior, food consumption, body and organ weights, hematological and biochemical values, macroscopic and microscopic findings on autopsy.


[Morphometric and immunohistochemical studies of the suprachiasmatic nucleus in the hereditary microphthalmic rat].

Morphometric and immunohistochemical analyses of the suprachiasmatic nucleus (SCN) were performed on hereditary microphthalmic rats. In normal rats, the number of cells and the volume of the SCN were 11, 631 and 6.7 x 10(-2) mm3 (an average taken from 12 SCNs). However, the neuronal population and volume of the SCN in hereditary microphthalmic rats were 7,450 and 4.5 x 10(-2) mm3 (an average taken from 14 SCNs), respectively. There were no significant differences in the size of neurons between normal and microphthalmic SCN neurons. Immunohistochemical studies showed that a considerable number of antivasopressin positive neurons were present in microphthalmic rats, despite their lack of the optic nerve. However, further detailed studies revealed that the number of antivasopressin positive neurons present in microphthalmic rats was only 68% of those found in normal rats. These findings suggest that the complete development of the SCN and vasopressin neurons depends on the visual input.


[Cryopreservation of unfertilized rat oocytes by ultrarapid freezing].

Unfertilized rat oocytes were placed in a highly concentrated solution of cryoprotectant (DAP 224:2M dimethylsulphoxide, 2M acetamide, 4M propylene glycol in PB1) in 0.5 ml sampling tubes and then immediately immersed into liquid nitrogen; thawing was conducted in a 37 degrees C waterbath. After thawing, 630 out of 968 oocytes (65.1%) were morphologically normal. After insemination in vitro of cryopreserved oocytes, the proportion of pronuclear oocytes with spermatail (s), male (s) and female pronuclei (8-10 h post insemination), and 2-cell embryos with two identical blastomeres (28-30 h post insemination) was 60.8% (152/250) and 29.8% (39/131), respectively. One hundred and fifty oocytes that were judged as pronuclear oocytes under the inverted microscope 8-10 h after insemination were transferred to the oviducts of pseudopregnant recipients; 18.7% (28/150) of the oocytes developed to normal young.


The domestication of Crocidura dsinezumi as a new laboratory animal.

The dsinezumi shrew (Crocidura dsinezumi), a small insectivore, has been bred for the first time as a laboratory animal. The original animals were captured using Sherman's live traps and transferred into wooden cages. After several generations they were housed in plastic cages. Their diet consisted of trout pellets, cat food, and water provided ad libitum. Monogamous pairs were housed together for 2-3 weeks for mating, and the male was separated from the female during delivery and nursing. In captivity, the reproductive activity was observed throughout the year and the gestation period was estimated at 28-30 days with a litter size of between 1 and 4 pups. Pups grew very rapidly, and reached adult body size (mean: male, 9.7 g; female, 8.3 g) and sexual maturation at 6-8 weeks of age. The reproductive life was estimated at one and a half years, while the longevity was approximately 2 years.