Capillary electrophoresis for pharmacokinetic studies

Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.     

嗜热脂肪芽孢杆菌DNA解链蛋白BstH2的纯化及性质研究

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀、硫酸铵分级及ＤＥＡＥ纤维素、磷酸纤维素、Ｂｌｕｅ－Ｓｅｐｈａｒｏｓｅ、ＦＰＬＣＭｏｎｏＱ、ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到了部分纯化ＤＮＡ解链蛋白ＢｓｔＨ２。ＢｓｔＨ２具有受ＤＮＡ促进的ＡＴＰ酶活力,没类型的核酸对ＢｓｔＨ２的ＡＴＰ酶活力的促进作用没。ＢｓｔＨ２在５５℃有最高ＡＴＰ酶活力。这种活力受大肠杆菌单链ＤＮＡ结合蛋白的抑制及随     

NUTRIENT LIMITATION OF BENTHIC ALGAE IN LAKE MICHIGAN: THE ROLE OF SILICA1

In the Laurentian Great Lakes, phytoplankton growth and biomass are secondarily limited by silica (Si), as a result of phosphorus (P) enrichment. Even modest levels of P enrichment can induce secondary Silimitation, which, in turn, promotes a shift from the native diatom phytoplankton flora to chlorophyte and cyanobacteria species. However, very little is known about the nutritional status of benthic populations and their response to nutrient enrichment. Two experiments were performed in the littoral zone of Lake Michigan where nutrients were delivered to in situ benthic algal (episammic and epilithic) assemblages using nutrient‐diffusing substrata. In order to test the hypothesis that benthic algae in Lake Michigan are Si limited, a 2 × 3 factorial experiment was used to deliver all combinations of Si, N, and P to resident assemblages growing on artificial substrata composed of natural (Si rich) versus calcium carbonate (Si poor) sand. A second experiment utilized a serial enrichment to evaluate the role of Si in mediating changes in taxonomic composition. These findings indicate that benthic algae in Lake Michigan exhibit signs of secondary Si limitation, and that their response to enrichment is similar to the phytoplankton. Moreover, natural sand substrata may provide a source of Si to resident benthic algae.     

MONITORING RAPID VALVE FORMATION IN THE PENNATE DIATOM NAVICULA SALINARUM (BACILLARIOPHYCEAE)1

After each division of a diatom cell, a new siliceous hypovalve is formed inside the silica deposition vesicle (SDV). We present the sequence of this early formation of the new valve in the pennate marine diatom Navicula salinarum (Grunow) Hustedt, visualized by using the fluorescent probe 2‐(4‐pyridyl)‐5‐((4‐(2‐dimethylaminoethylamino‐carbamoyl)methoxy)phenyl)oxazole (PDMPO). Our observations confirm that two‐dimensional expansion of the growing valve is a rapid process of no more than 15 min; three‐dimensional completion of the valve appears to be slower, lasting most of the time valve formation takes. The results are relevant to studies of the timing of molecular processes involved in valve formation (i.e. the bio‐ and morphogenesis of the SDV) in relation to uptake and transport of silicic acid. Use of this probe helps us to identify specific developmental stages for further detail analysis of diatom basilica formation, which eventually could lead to obtaining enriched SDV fractions.     

CELL DIVISION AND MORPHOGENESIS OF THE CENTRIC DIATOM CHAETOCEROS DECIPIENS (BACILLARIOPHYCEAE) II. ELECTRON MICROSCOPY AND A NEW PARADIGM FOR TIP GROWTH

Valve morphogenesis starts when the silica deposition vesicle (SDV) expands across a cleavage furrow covered by an unidentified layer, which may aid in its shaping. A labiate process (LP) is present only in the outer valve of terminal cells in the filament. Before these particular cells form setae, a layered "labiate process apparatus" (LPA) appears on the SDV in the exact center of the forming valve, near the microtubule center arising after cleavage. The LPA thereafter surmounts the lips of the LP as it forms. After the girdle bands separate slightly, two lateral protrusions develop in the corners of the cell. These nascent setae are lined internally by a cylindrical, fibrous band (sleeve), which assembles immediately ahead of the expanding edge of the SDV, very close to the plasmalemma. Then these protrusions, lined by the fibrous band, the SDV, and the forming silica wall, grow through two gaps in the girdle bands. The cytoplasm at the tip of the growing seta is naked. Immediately behind the tip, this fibrous band is adpressed to the plasmalemma and thereby apparently defines the diameter of the seta; it extends to internally ensheath the tipmost edge of the SDV for a short distance, like a tight-fitting inner sleeve. This structure is considered the major organelle involved in seta morphogenesis. Microtubules (MTs), while present, are variable in extent and disposition within the seta. Turgor pressure is considered irrelevant in driving seta growth. Instead, a new paradigm proposed for tip-growing cells generally, may apply to seta morphogenesis, as follows. If, as is suspected, the fibrous band contains actin, cycling of this actin (as in animal cells undergoing ruffling or filopodial extension) could drive seta extension via attachment of the band to the just-formed silica wall. The band is visualized as a molecular treadmill whose support base, the new wall, is being continually extended; extension is controlled and generated strictly at the tip.     

Morphometrische Befunde zum submikroskopischen Aufbau des Sehnerven der Ratte

Zusammenfassung An 10 normalen Sehnerven (Länge: 10 mm; Durchmesser: 0,5 mm) erwachsener Albino-Ratten wurden morphometrische licht- und elektronenmikroskopische Untersuchungen mit der Treffermethode an 3 Stellen durchgeführt: 1 mm vor dem Bulbus oculi (Meßort A); Mitte zwischen Bulbus und Chiasma (Meßort B); 1 mm vor dem Chiasma opticum (Meßort C). Die statistisch signifikanten Ergebnisse der Untersuchung sind: 1. die Gesamtzahl der markhaltigen Fasern im Sehnerven der erwachsenen Ratte beträgt in elektronenmikroskopischen Aufnahmen 107152±6780. 2. Der relative Volumenanteil der Markfasern (Axone und Markscheiden) nimmt von A über B nach C um 13,3 Vol.- % zu, der relative Volumenanteil des Interstitiums (Glia, Gefäßmesenchym und extracellulärer Raum) um den gleichen Betrag ab. 3. Mit der Volumenabnahme des Interstitiums korreliert eine Abnahme der Gliakernzahl pro mm³ von A über B nach C um 46,2% und eine Abnahme der Dichte des Capillarnetzes gemessen an der Zahl der Endothel- und Pericytenkerne. 4. Die Größe der Querschnittsflächen durch den Sehnerven ist am Meßort B (Mittelabschnitt) kleiner als bei A und C. 5. Die Berechnung der absoluten Volumina aus den relativen Volumenwerten und der Querschnittsflächengröße ergibt, daß das Volumen des Interstitiums zwischen A und B um 34,8% abnimmt, während das Markfaservolumen hier fast unverändert bleibt. Dagegen nimmt zwischen B und C das Markfaservolumen um 11,8% zu, während das Volumen des Interstitiums weiter, aber nur geringfügig abnimmt. Die Ergebnisse zeigen, daß die Umfang. zunahme des Sehnerven vom Mittelabschnitt (Durchtritt durch den Canalis opticus) gegen beide Endabschnitte durch Volumenvermehrung jeweils verschiedener Gewebskomponenten bedingt ist.

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Stereological study on the submicroscopic structure of the rat optic nerve

Summary The normal optic nerves of ten adult albino rats were studied using stereological methods of cell counting in light microscopic preparations and point-hit counting on electron micrographs. The observations were confined to three locations of the nerve: one mm from the eyeball (point A), one mm from the optic chiasm (point C) and midway between these sites corresponding to the canalis opticus (point B). The statistically significant results are: 1. the total number of myelinated nerve fibres in the optic nerve is 107152±6780. 2. The volume of myelinated nerve fibres (axons plus myelin sheaths) increases by 13.3% from point A to C, whereas the volume of interstitium (glial cells, capillaries and extracellular space) decreases concomitantly. 3. The decrease in interstitial tissue corresponds to a 46.2% decrease in glial cells between A and C and of capillaries indicated by the numbers of endothelial cells and pericytes. 4. The surface area of cross sections of the optic nerve at point B is smaller than at point A and point C. 5. The absolute volume of the two structural components in the optic nerve, calculated from percentage volume and cross section surfaces, showed that the volume of interstitial tissue decreases from the eyeball to the intermediate portion by 34.8% whereas the volume of myelinated fibres remains nearly constant. From the intermediate portion to the optic chiasm the volume of myelinated fibres increases by 11.8% whereas the interstitial volume further decreases slightly. Thus the increasing diameter of the optic nerve from the middle part to both ends is caused by increases in volume of different tissue constituents.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Zur Kenntnis der terminalen Strombahn im Myocard von Ratte und Katze

Zusammenfassung Das Verzweigungsmuster der terminalen Strombahn des Myocards wird an Zupfpräparaten von 67 Ratten- bzw. Katzenherzen untersucht. Die Gefäße wurden mit Farbsuspensionen gefüllt. Die Schwierigkeiten der vollständigen Darstellung des Kapillarbettes werden diskutiert. Durch aufeinanderfolgende Injektion mit mehreren Farben gelingt es, den arteriellen und venösen Kapillarschenkel unterschiedlich darzustellen. Zur genauen Analyse werden von einzelnen Muskelblättchen ausgedehnte Gefäßkarten hergestellt. Folgende Befunde wurden erhoben: Als feinste Zuleitungsgefäße münden die Präkapillaren in das Kapillarnetz. Ihre Wand enthält nur vereinzelte Muskelzellen. Die Präkapillaren verlaufen parallel oder senkrecht zu den Muskelfasern und teilen sich in gleichwertige Äste (dichotom) oder geben jeweils als Stammgefäß seitliche Äste ab. Die Arteriolen bzw. Präkapillaren anastomosieren nicht miteinander.Die gewöhnlich weiten Venolen bestehen im wesentlichen nur aus einem Endothelrohr. Sie verlaufen zunächst senkrecht zur Muskulatur und münden in abführende, meist längsgerichtete Venen ein. Anastomosen unter den Venolen sind selten.Zwischen Präkapillaren und Venolen sind Kapillarbahnen ausgespannt. Die Kapillaren verlaufen parallel zueinander im Abstand einer Muskelzellbreite und stehen über Queranastomosen miteinander in Verbindung.Die Kapillarwege von Präkapillaren zu Venolen variieren zwischen 100 m und 800 m. Messungen an zwei Herzen ergaben eine mittlere Strecke von 310 m (Ratte) bzw. 400 m (Katze).Die mittlere unverzweigte Kapillarstrecke beträgt im etwas kontrahierten Rattenherzen 65 m, im mäßig erschlafften Katzenherzen 110 m. Da sich höchstens 1/3 der Kapillaräste untereinander zu Maschen verbinden, entsteht ein sehr lockeres Netzwerk.Da benachbarte Kapillaren zu gleichen oder verschiedenen Präkapillaren und Venolen gehören können, sind mehrere Gefäanordnungen im Myocard unterscheidbar:Selten sind reine Gleichstromanordnungen (vgl. Krogh, 1918/19) oder Gegenstromanordnungen (vgl. Diemer, 1965) zu beobachten, bei denen die Kapillaren am gleichen Ort anfangen bzw. enden und benachbarte Kapillaren in gleicher oder entgegengesetzter Richtung durchströmt werden.Überwiegend finden sich asymmetrische Kapillaranordnungen (vgl. Grunewald und Lübbers, 1966), bei denen Kapillaranfänge bzw. -enden gegeneinander versetzt sind und benachbarte Kapillarabschnitte in gleicher oder entgegengesetzter Richtung durchströmt werden. Die morphologischen Befunde zeigen, daß physiologischen Untersuchungen ein gemischtes Versorgungsmodell für den Herzmuskel (Grunewald und Lübbers, 1966) zugrunde gelegt werden kann.

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The microcirculatory bed in the myocardium of the rat and the cat

Summary The branching modus of the microcirculatory bed is studied in isolated musclelayers from 67 hearts of rats or cats. The vessels are filled with different dye-suspensions. The methods to demonstrate the capillary bed by perfusion are discussed. It is shown that several dyes injected consecutively can mark the arterial and venous part of the capillary with different colours. For exact analysis maps are prepared of extensive parts of the microcirculatory bed.The precapillaries, the smallest vessels of the distributing system, are provided with only sporadic muscle cells. They run parallel to the muscle fibers or they cross them. They either divide into equal branches in a dichotomous manner or extend as trunk-vessels, from which the capillaries originate as branches. The arterioles and precapillaries don't anastomose with each other. The venous drainage begins with venoles which usually consist of only a wide endothelial tube. They cross the muscle fibers and open into veins which usually extend in the direction of the muscle layers. Anastomoses among the venoles are rare.Between precapillaries and venoles the capillary ways extend. The capillaries run parallel to each other in a distance of one muscle cell. They are connected by short transversal anastomoses. The length of the capillary way between precapillaries and venoles varies between 100 m and 800 m. According to measurements in two hearts there follows an average way of 310 m (rat) or 400 m (cat). The average length of the unbranched capillary segments was 65 m (rat) or 110 m (cat). As at most one third of the capillary branches are combined to mashes there is only a loose capillary-network.Adjacent capillaries may belong to the same or to different precapillaries and venoles, so that there can be differentiated between several different patterns of the microcirculatory bed:There are seldom found pure arrangements in such a way, that adjacent capillaries begin or end at the same place and that the blood flows in equal or contrary directions (cf. Krogh, 1918/19; Diemer, 1965).Mostly asymmetric arrangements are found with capillaries beginning or ending at staggered places and with equal or contrary flow directions in the capillary segments. The morphological results show that the base for physiological studies of the nutrition of the myocardium ought to be a mixed model of the capillary bed.

Die Arbeit entstand unter der Leitung von Herrn Prof. Dr. E. Lindner, Regensburg.     

A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome

While macroalgal microbiomes are the focus of many recent studies, there is little information about microbial spatial diversity across the thallus. Reliance on field material makes it difficult to discern whether recovered microbiomes belong to the host or its epiphytes, and technical comparisons of macroalgal samples for microbial studies are needed. Here, we use a common garden approach that avoids the problem of epiphytes, particularly at holdfasts, to examine the microbiome of Porphyra umbilicalis (strain Pum1). We used the V6 hypervariable region of the 16S rDNA with Illumina HiSeq sequencing and developed PNA clamps to block recovery of organelle V6 sequences. The common garden approach allowed us to determine differences in the microbiome at the holdfast versus blade margin. We found a notable increase in the relative abundance of Planctomycetes and Alphaproteobacteria at the holdfast, particularly of the possible symbiont Sulfitobacter sp. Nonadjacent 1.5 cm² samples of blade margin had microbiomes that were not statistically different. The most abundant phylum in the overall microbiome was Proteobacteria, followed by Bacteroidetes. Because phycologists often work in remote sites, we compared three stabilization and preparation techniques and found silica gel desiccation/bead‐beating and flash‐freezing/lyophilization/bead‐beating to be interchangeable. Core taxa (≥0.1% of sequences) across treatments were similar and accounted for ≥95% of all sequences. Finally, statistical conclusions for all comparisons were the same, regardless of which microbial community analysis tool was used: mothur or minimum entropy decomposition.     

Sequencing of Gel-Isolated Proteins Using Microblotter Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS–PAGE at low picomole levels can be analyzed by the new integrated system.