Minor structural modifications convert a selective PPARalpha agonist into a potent, highly selective PPARdelta agonist

We report the solid-phase synthesis and pharmacological evaluation of a new series of small-molecule agonists of the human peroxisome proliferator-activated receptor delta (PPARdelta) based on a lead structure from our PPARalpha program. Compound 33 showed good pharmacokinetics.     

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

Vergleichende Histologische Untersuchungen anPolyergus rufescens Latr. undRaptiformica sanguinea Latr.

Zusammenfassung Die obligatorisch dulotische AmeisenartPolyergus rufescens Latr. (P. r.) wird histologisch verglichen mit der nur fakultativ sozial-parasitischenRaptiformica sanguinea Latr. (R. s.). In den Rectalpapillen beider Arten findet sich zwischen Innen- und Aussenschicht eine Zone mit Granulae, die auf Plasmafäden aufgereiht sind. Ein Teil der Drüsen vonP. r. weist Degenerationen auf: Epinotaldrüsen (alte Bezeichnung: Metathorakaldrüsen) fehlen ganz, die Labialdrüsen sind weitgehend reduziert, die Propharynxdrüsen (Maxillardrüsen) haben weniger Einzelzellen als die vonR. s. Mandibel- und Postpharynxdrüsen (Pharyngealdrüsen) zeigen keine Reduktionen. Die Maxillendrüsen («Zungendrüsen» nachOtto, 1958) werden genau beschrieben und abgebildet. Ihre Einzelzellen sind beiP. r. zahlreicher als beiR. s. Einzelne Drüsenzellen kommen nahe der Cuticula verstreut vor, besonders bei Gelenkmembranen. Die Nebendrüsen beider Arten zeigen einschneidende altersmässige Veränderungen, die als Uebergang der sezernierenden Phase in eine speichernde angesehen werden. In den Oberflächenabmessungen der Corpora pedunculata, die als besonders leistungsfähige Assoziationszentren angesehen werden, istR. s. nur wenig überlegen. Von einer Degeneration beiP. r. kann diesbezüglich keine Rede sein. Bei einem soeben geschlüpftenR.s.- wurden im Prothorax nierenförmige Organe gefunden, die einen histologischen Abbau zeigten; es handelt sich vermutlich um die inkretorisch wirksamen Prothorakaldrüsen. Eine Ueberprüfung der Fühlersinnesorgane ergab keine wesentliche Unterlegenheit vonP. r. gegenüberR. s. Bei den Geschlechtsorganen der wurde vor allem der noch wenig untersuchte Ductus ejaculatorius mit Sperrkeil sowie die Penisblase beschrieben und abgebildet. Der Entwicklungszyklus der -Ovariolen nachWeyer (1927) beiFormica rufa liegt auch beiR. s. vor, tritt bei jedoch nur in abgeschwächter form auf. Im Kernchromatin der meisten Organe lässt sich eine zunehmende Bildung von Sammelchromozentren bei fortschreitendem Alter feststellen.R. s. hat dabei vom Schlüpfen an einen «Vorsprung», der auch bei älteren Tieren bestehen bleibt. Nach der Terminologie vonWhite (1943, 1945) istP. r. als mikroheterochromatisch,R. s. als megaheterochromatisch zu bezeichnen. Mögliche und wahrscheinliche Beziehungen zwischen Sozialparasitismus und Histologie der untersuchten Arten werden erörtert. Ausserdem werden die neuen histologischen Befunde diskutiert.

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Summary The compulsory dulotic ant speciesPolyergus rufescens L. (P. r.) is being compared with the only facultative social-parasiticRaptiformica sanguinea Latr. (R. s.) with regard to their histology. Some of the glands ofP. r. show degenerations. Epinotal Glands are missing at all, Labial Glands are reduced, Propharyngeal glands (Maxillar Gl.) have fewer single cells than those ofR. s. Mandibular and Postpharyngeal (Pharyngeal) Glands show no reductions. The Maxillar Glands («Tongue Glands» according toOtto, 1958) are exactly described and illustrated. Those ofP. r. have more single cells than those ofR. s. Single gland cells are found scattered near the cuticula, especially at membranes of joints. The «Nebendrüsen» according toForel (glands beside the poison bladder) show enormous age-conditioned variations, which are interpreted as a transitional stage of a phase of secretion and a hoarding phase. the measures concerning the surface of the Corpora pedunculata ofR. s., which are considered as especially effectful association-centers, are only a bit larger than those ofP. r. There could not be found any symptom of degeneration of brain. In aR. s.-, just hatched, kidney-shaped organs were found in a state of histological reduction. These are supposed to be incretoric Prothoracal Glands. An examination of the antennae sense-organs stated no essential deficiency ofP. r. In the male genital organs the little examined ductus ejaculatorius with the close wedge («Sperrkeil») is described and illustrated as well as the penis-bladder. The development-cyclus of the -ovarioles according toWeyer (1927) inFormica rufa is also found inR. s., but inP. r. only in an attenuated way. In the nuclear chromatine of the most organs an increasing formation of compact chromocentres in the progress of age is stated. This tendency is at any time more distinct inR. s., even in old individuals. According toWhite (1943, 1945)P. r. is microheterochromatic andR. s. megaheterochromatic. Relations between social parasitism and histology of the present ant species are discussed.

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Résumé La fourmi dulotique obligatoirePolyergus rufescens (P. r.) est comparée histologiquement avec laRaptiformica sanguinea Latr. (R. s.) seulement facultativement social-parasitique. Dans les papilles rectales des deux espèces se trouve une zone avec Granulae, rangée sur fils de plasma. Une part des glandes deP. r. montre une dégénérescence: les glandes épinotales (glandes métathoraciques) manquent totalement, les glandes labiales sont diminuées largement, les glandes propharyngeales (gl. maxillaires) ont moins de cellules isolées que celles desR. s., les glandes mandibulaires et postpharyngeales (pharyngeales) ne montrent pas des dégénérescences. Les glandes maxillaires («glandes linguales», conformément àOtto, 1958) sont exactement décrites et illustrées. Leurs cellules isolées deP. r. sont plus nombreuses, comme celles deR. s. Quelques cellules glandulaires se trouvent dispersées près de la cuticule, spécialement près de la membrane articulée. Les glandes supplémentaires des deux espèces montrent des variations énormes dépendant de l'âge, qui sont regardées comme une transition de la phase de la sécrétion à une phase d'emmagasinage. En ce qui concerne les mesures de surface des corpora pedunculata qui sont regardés comme des centres particulièrement efficaces,R. s. est seulement un peu surclassée. On ne peut pas parler d'une dégénérescence deP. r. à cet égard. On a trouvé des organes en forme de reins dans le Prothorax desR. s.- écloses à l'instant. Ces organes montraient une dégradation histologique. Il s'agit probablement de glandes prothoraciques de sécrétion intérieure. Après examen des organes sensoriels des antennes on ne trouvait pas une infériorité essentielle deP. r. en comparaison deR. s. Dans les organes génitaux des , le Ductus éjaculatorius avec le dispositif d'accrochage peu connu ainsi que la bulle du pénis sont décrits et illustrés avant tout. Le cycle d'évolution des ovarioles, conformément àWeyer (1927) (Formica rufa), est aussi visible auprès deR. s., mais il se montre dans une forme diminuée seulement auprès des . Dans le noyau chromatique de la plupart des organes on peut voir un développement montant des chromo-centres collectifs durant le vieillissement. Dans ce cas,R. s. a une avance à partir de l'éclosion, qui reste aussi chez les animaux plus âgés. Conformément à la terminologie deWhite (1943, 1945),P. r. est désignée comme microhétérochromatique etR. s. comme mégahétérochromatique. Des relations possibles et probables entre le parasitisme social et l'histologie des espèces étudiées sont discutées; en outre, les nouvelles constatations histologiques sont discutées.

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Herrn Professor Dr.Gösswald bin ich für die Anregung zu dieser Arbeit und seine grosszügige Unterstützung zu grossem Dank verpflichtet.     

The carcinogenic effect of TPA (12-0-tetradecanoylphorbol-13-acetate) when applied to the skin of hairless mice

Groups of hairless mice received one, two, five and fifty applications of 20 nmoles TPA (12-0-tetradecanoylphorbol-13-acetate) on the skin of the back, and were observed for 20 months. The animals developed some papillomas, some squamous cell carcinomas, some fibrosarcomas of the dermis, and some malignant and benign tumours in internal organs. There was a small, not significantly different, incidence of benign and malignant tumours after 1, 2 and 5 paintings, and a significantly higher tumour incidence after 50 applications. Apart from reticuloses, which are commonly seen in these animals, the occurrence of other tumours is believed to be related to the TPA treatment. The results are interpreted as showing that TPA, like croton oil, should be regarded as a complete carcinogen.     

Proteomic analysis of the Simkania‐containing vacuole: the central role of retrograde transport

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane‐bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania‐containing vacuole (SnCV) is a critical host–pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER‐SnCV‐membranes using label free LC‐MS² to define the pathogen‐containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome‐to‐trans‐Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome‐to‐TGN transport for nutrient acquisition and growth.     

Substrate Degradation Kinetics, Microbial Diversity, and Current Efficiency of Microbial Fuel Cells Supplied with Marine Plankton

The decomposition of marine plankton in two-chamber, seawater-filled microbial fuel cells (MFCs) has been investigated and related to resulting chemical changes, electrode potentials, current efficiencies, and microbial diversity. Six experiments were run at various discharge potentials, and a seventh served as an open-circuit control. The plankton consisted of a mixture of freshly captured phytoplankton and zooplankton (0.21 to 1 mm) added at an initial batch concentration of 27.5 mmol liter⁻¹ particulate organic carbon (OC). After 56.7 days, between 19.6 and 22.2% of the initial OC remained, sulfate reduction coupled to OC oxidation accounted for the majority of the OC that was degraded, and current efficiencies (of the active MFCs) were between 11.3 and 15.5%. In the open-circuit control cell, anaerobic plankton decomposition (as quantified by the decrease in total OC) could be modeled by three terms: two first-order reaction rate expressions (0.79 day⁻¹ and 0.037 day⁻¹, at 15°C) and one constant, no-reaction term (representing 10.6% of the initial OC). However, in each active MFC, decomposition rates increased during the third week, lagging just behind periods of peak electricity generation. We interpret these decomposition rate changes to have been due primarily to the metabolic activity of sulfur-reducing microorganisms at the anode, a finding consistent with the electrochemical oxidization of sulfide to elemental sulfur and the elimination of inhibitory effects of dissolved sulfide. Representative phylotypes, found to be associated with anodes, were allied with Delta-, Epsilon-, and Gammaproteobacteria as well as the Flavobacterium-Cytophaga-Bacteroides and Fusobacteria. Based upon these results, we posit that higher current efficiencies can be achieved by optimizing plankton-fed MFCs for direct electron transfer from organic matter to electrodes, including microbial precolonization of high-surface-area electrodes and pulsed flowthrough additions of biomass.     

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.