PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA,a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.     

Die durch Argas (Persicargas) persicus-Larven bedingte Paralyse der Hühner

Zusammenfassung Bei der durch Argas (Persicargas) persicus-Larven bedingten Zeckenparalyse der Hühner handelt es sich um eine generalisierte Affektion des peripheren Nervensystems, wobei es zu einer funktionellen Schädigung der schnell leitenden Nervenfasern kommt.Bei 12 Tieren mit schwersten Paralysen waren die maximalen motorischen Nervenleitungsgeschwindigkeiten, bei gleichzeitig erforderlicher Erhöhung der motorischen Schwellen- bzw. Supramaximalreize, auf 55% herabgesetzt sowie die Reizantwortpotentiale aus der distalen Muskulatur auf 15–30% abgefallen. Die distalen Überleitungszeiten verlängerten sich dabei nur geringfügig. Bei repetitiver distaler Nervenreizung kam es zu einem deutlichen Amplitudenabfall. 3 Tage nach klinischer Restitution hatten sich die neurophysiologischen Veränderungen noch nicht zurückgebildet.Bei 8 Tieren mit mittelgradigen Paresen war die Schädigung des peripheren Nervensystems nicht so stark ausgeprägt. Die maximalen motorischen Nervenleitungsgeschwindigkeiten waren nur auf 70% vermindert, während die motorischen Schwellen- und Supramaximalreize leicht erhöht waren. Die Werte für die Amplituden der Summenpotentiale waren im Vergleich mit den schwerst gelähmten Tieren bedeutend größer, die distalen Latenzzeiten blieben dagegen unverändert. Bei repetitiver Nervenreizung verminderten sich die Amplituden der Antwortpotentiale nur geringfügig. 3 Tage nach der klinischen Rekonvaleszenz waren bei allen Tieren die neurophysiologischen Veränderungen, bis auf eine noch bestehende Verminderung der Summenpotentiale, wieder völlig zurückgebildet.

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Tick paralysis in chickens caused by Argas (Persicargas) persicus-Larvae

Summary In tick paralysis caused by Argas (Persicargas) persicus-larvae a generalized affection of peripheral nervous system especially of fast conducting nerve fibres was found.In 12 animals with severe paralysis maximal motor conduction velocity slowed down to 55% of the initial value and the amplitudes of evoked compound muscle action potentials decreased to 15–30% of the initial value. Simultaneously motor threshold resp. supramaximal stimuli to the nerves were increased. Distal latencies changed only a little. By repetitive distal nerve stimulation muscle action potential amplitudes showed a decrement. Three days after clinical restitution neurophysiologically an affection of peripheral nerves to a small extent could be shown. The affection of peripheral nerves in 8 animals with moderate pareses was less pronounced. The reduction of motor conduction velocity went down only to 70% of the initial value whereas threshold and supramaximal stimuli were only slightly increased. There were in comparison with the severe paralyzed animals higher amplitudes of evoked compound muscle action potentials. Distal latencies remained unchanged. By repetitive nerve stimulation only a small decrement of muscle action potential amplitudes could be stated. Three days after clinical restitution neurophysiologically only a slight reduction of the amplitudes of the evoked muscle action potentials could be measured.

     

Das Verhalten von Blutdruck und Nierendurchblutung während des Carotissinus-Reflexes am wachen Hund

Zusammenfassung An 15 Bastardhunden wurde der Einfluß einer Na-Pentobarbital-Narkose sowei einer zusätzlichen chirurgischen Präparation auf die Ruhewerte von Blutdruck und Nierendurchblutung und deren Änderung nach doppelseitigem Carotisverschluß im Vergleich zum wachen Tier untersucht.Barbiturat-Injektion (30 mg/kg i.v.) allein erhöhte die Herzfrequenz, den systolischen und diastolischen Blutdruck, änderte jedoch nicht die mittlere Nierendurchblutung. Zusätzliche chirurgische Präparation verstärkte diese Veränderungen mit Ausnahme vom systolischen Blutdruck und der Nierendurchblutung.Bei Carotisverschuluß kam es im steady state zu einer vermehrten reflektorischen Herzfrequenzsteigerung nach Barbiturat-Injektion sowie nach Barbiturat mit chirurgischer Präparation. In Barbiturat-Narkose stieg der mittlere Blutdruck stärker an als am wachen Tier; diese Veränderung war nach chirurgischer Präparation noch deutlicher ausgeprägt. Die mittlere Nierendurchblutung wurde im steady state in keiner der 3 Gruppen signifikant verändert. Der reflektorische Herzfrequenz- und Blutdruckanstieg erfolgte im Wachzustand rascher als nach Barbiturat und Barbiturat mit chirurgischem Trauma. Unter letzteren Bedingungen fehlte auch das im Wachzustand beobachtete Unterschießen der Herzfrequenz nach Öffnen der Carotismanschetten. Der druckpassive overshoot der Nierendurchblutung am wachen Tier fehlte sowohl nach Barbiturat als auch nach zusätzlicher chirurgischer Präparation.Die Ergebnisse zeigen, daß der mit Na-Pentobarbitural narkotisierte und der narkotisierte, akut operierte Hund gegenüber dem Wachzustand betreffend der Ruhewerte und der reflektorischen Veränderungen der Herzfrequenz und des Blutdrucks im steady state des Carotissinus-Reflexes zwei quantitativ unterschiedlich reagierende Kreilaufpräparate liefert. Gemessen an der Beseitigung sowohl der raschen Herzfrequenz- und Blutdruckveränderungen als auch des phasischen Einschwingvorganges der Nierendurchblutung durch Narkose und Narkose mit Trauma unterscheiden sich solche Präparate auch qualitativ vom Wachzustand.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Cerebrovascular reactivity to hypercapnia is unimpaired in breath-hold divers

Hypercapnic cerebrovascular reactivity is decreased in obstructive sleep apnoea and congestive heart disease perhaps as a result of repeated apnoeas. To test the hypothesis that repeated apnoeas blunt cerebrovascular reactivity to hypercapnia, we studied breath hold divers and determined cerebrovascular reactivity by measuring changes in middle cerebral artery velocity (MCAV, cm s⁻¹) per mmHg change in end-tidal partial pressure of CO₂ (     ) in response to two hyperoxic hypercapnia rebreathing manoeuvres (modified Read protocol) in elite breath-hold divers (BHD, n = 7) and non-divers (ND, n = 7). In addition, ventilation and central (beat-to-beat stroke volume measurement with Modelflow technique) haemodynamics were determined. Ventilatory responses to hypercapnia were blunted in BHD versus ND largely due to lower breathing frequency. Cerebrovascular reactivity did not differ between groups (3.7 ± 1.4 versus 3.4 ± 1.3% mmHg⁻¹     in BHD and ND, respectively; P = 0.90) and the same was found for cerebral vascular resistance and MCAV recovery to baseline after termination of the CO₂ challenge. Cardiovascular parameters were not changed significantly during rebreathing in either group, except for a small increase in mean arterial pressure for both groups. Our findings indicate that the regulation of the cerebral circulation in response to hypercapnia is intact in elite breath-hold divers, potentially as a protective mechanism against the chronic intermittent cerebral hypoxia and/or hypercapnia that occurs during breath-hold diving. These data also suggest that factors other than repeated apnoeas contribute to the blunting of cerebrovascular reactivity in conditions like sleep apnoea.     

Applications of a rat multiple tissue gene expression data set

With the sequencing and assembly of the rat genome comes the difficult task of assigning functions to genes. Tissue localization of gene expression gives some information about the potential role of a gene in physiology. Various examples of the utility of multiple tissue gene expression data sets are illustrated here. First, we highlight their use in finding genes that might play an important role in a particular tissue on the basis of exclusive expression in that tissue or coexpression with a gene or genes with known function. Second, we show how this data might be used to explain known phenotypic differences between strains. Third, we show how expression patterns of genes in a genomic interval might identify candidate genes in quantitative trait loci (QTL) mapping studies. Lastly, we show how multiple tissue and species data can help researchers prioritize follow up studies to microarray experiments. All of these applications of multiple tissue gene expression data sets will play a role in functionally annotating the rat genome.     

Untersuchungen über die Entwicklung und den Infektionsverlauf von Aegyptianella pullorum Carpano, 1928, im Huhn

Zusammenfassung 1. Aegyptianella pullorum nimmt im allgemeinen in Hühnern, unabhängig vom Infektionsweg, einen typischen Verlauf. Die Entwicklung des Parasiten vollzieht sich in den Erythrozyten des Wirtstieres in drei Phasen, die aber nur auf Grund typischer morphologischer Merkmale unterscheidbar sind und sich in der zeitlichen Reihenfolge nicht trennen lassen.2. Durch die Übertragung des Blutes von Kücken, die durch Zeckenbisse infiziert wurden, auf empfängliche Tiere in bestimmten Zeitabständen nach dem Saugakt infektiöser Argas (Persicargas) persicus-Zecken sowie auf Grund histologischer Untersuchungen infizierter Kücken kann eine präerythrozytäre Phase in der Entwicklung von Aegyptianella pullorum ausgeschlossen werden.3. Die in weißen Blutzellen, den Kupfferschen Sternzellen der Leber und frei im Blutplasma gefundenen Parasiten werden nicht als exoerythrozytäre Entwicklungsstadien von Aegyptianella pullorum interpretiert.

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Investigations into the development and the course of infection of aegyptianella pullorum Carpano, 1928, in chicken

Summary Generally Aegyptianella pullorum takes a typical course in chicken independent from the route of infection. In the erythrocytes of vertebrate hosts, the development of the parasite passes through three phases, which can be differentiated only by the typical morphological features.By transmission of blood from naturally infected chicken to susceptible animals, after engorgement of infected Argas (Persicargas) persicus, as well as by histological investigations of infected chicken, a preerythrocytical phase in the development of Aegyptianella pullorum can be excluded.The parasites, found in white blood cells, in the Kupffer cells of the liver and free in the blood plasma, are not interpreted as exoerythrocytic developmental stages of Aegyptianella pullorum.

     

Antibiotic prophylaxis influences cardiovascular stability in complicated surgery

Objective:Antibiotic prophylaxis is used in many surgical procedures but there are frequent cardiovascular instabilities following antibiotics in perioperative period. A clinic modelling randomised trial (CMRT) in pigs was developed to compare the effects of 2 commonly used antibiotic combinations on cardiovascular stability during major surgery. Materials and methods:Thirty pigs (both sexes) were randomised into 3 groups, receiving either saline (placebo), co-amoxiclav or cefuroxime/metronidazole in clinically relevant doses as antibiotic prophylaxis. A laparotomy was performed and the abdomen remained open. Surgical complications were simulated by removing one third of the blood volume. For fluid resuscitation, 500 ml hetastarch (HAES^TM) were infused rapidly (therapy of complication) and polymyxin B (15 mg/kg bodyweight) was applied for induction of histamine release reactions (complication of therapy). The main end points were histamine release reactions, these were classified by 2 blinded investigators. Results:Neither cardiovascular changes nor histamine release reactions were detected immediately after the administration of antibiotics or placebo alone. Plasma histamine concentrations increased after bleeding in the co-amoxiclav group (p < 0.05). After fluid resuscitation and induction of anaphylactoid reactions, the median histamine release and cardiovascular changes were not significantly different between the groups. However, the incidence of typical histamine release related reactions differed significantly between the groups: 8/10 for the controls, 6/10 in the co-amoxiclav and 2/10 in the cefuroxime/metronidazole group (p < 0.05). Conclusions:The stability and reproducibility of this model clearly demonstrated the concept of a ‘clinic modelling randomised trial’ as a useful tool. Antibiotic prophylaxis influences the organism’s capability to cope with intraoperative bleeding and fluid resuscitation problems. Indeed antibiotic prophylaxis may be beneficial. These effects of antibiotics could only be demonstrated in complex surgical models. Thus new antibiotics should be investigated in complex animal models prior to prospective randomised clinical trials or usage in clinical practice.     

Separation of mycobacterial soluble antigens by isoelectric focusing

Ion-exchange chromatography and gel filtration have been reported to yield partial separation of mycobacterial antigens. These procedures were used in combination with isoelectric focusing in an attempt to purify antigens of Mycobacterium tuberculosis strain H37Ra. The fractionating action of isoelectric focusing is dependent upon differences in the isoelectric points of the proteins to be separated. Culture filtrate of M. tuberculosis H37Ra was chromatographed on Sephadex G-200. This resulted in two widely separated peaks. The first peak, presumably containing high-molecular-weight substances, was then fractionated on a diethylaminoethyl Sephadex anion-exchange column. Three peaks were collected, and each was subjected to isoelectric focusing. Each peak was further separated into two or more fractions. The serological reactivity of each fraction was determined by immunodiffusion and immunoelectrophoresis. Sensitized guinea pigs were also skin-tested with the fractions. Two of the fractions contained only a single precipitinogen. One fraction contained two precipitinogens. A fourth fraction contained three precipitinogens and was also the only fraction to display sensitin activity. Four of the fractions were inactive either as precipitinogens or sensitins. The results suggest that the methods described are useful for the separation of mycobacterial antigens.     

Comparison of three methods to measure absolute cerebral hemoglobin concentration in neonates by near-infrared spectrophotometry

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in micromol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1-36.4) weeks, birthweight of 1386 (680-2820) g and a postnatal age of 2.5 (0.5-6) days, who needed supplemental oxygen, were enrolled. The mean+/-standard deviation tHbg was 150.2+/-41.8 micromol/L (range of 61.6-228.9 micromol/L), the tHbo was 62.1+/-27.2 micromol/L (26.0-110.8 micromol/L) and the tHbgo was 89.3+/-45.6 micromol/L (26.5-195.9 micromol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow's C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r(2)=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r(2)=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.