Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

Inhibitory action of a conditioning procedure on visual responsive neurons of the nucleus reticularis thalami in rats

Summary In urethane anesthetized rats neuronal responses of the visual part of nucleus reticularis thalami (vTR) to light were compared with those during pairing light as a conditioned stimulus (CS) with the electrical stimulation of the rat's tail (US). The intensity of the US was adjusted to the minimum required to evoke a slight freezing behavior in the awake rat. The firing rate of most vTR neurons decreased in the period between light and US application (P < 0.01). Significant response modulations to light were observed in 39% of the units, in most of them they persisted over an extinction period of 15 min. In addition, neurons which were predominantly inhibited by conditioning sometimes changed from regular spiking to a burst pattern. The results support the hypothesis that conditioning related facilitation of geniculate neurons observed in previous experiments can be explained at least partly by disinhibition of geniculate units from vTR.     

Wirkung des Äthylazetatextraktes von Chrysanthemum morifolium auf Plasmodium falciparumIn Vitro

Zusammenfassung Die Wirkung des ?thylazetatextraktes von Chrysanthemum morifolium auf P. falciparum wurde in vitro untersucht. W?hrend sich die Parasit?mie bei der Kontrollgruppe bis zu 10. 90 % entwickelte, wurde bei dem mit ?thylazetatextrakt in einer Konzentration von 100 μg/ml Medium am 5. Tag der Kultur eine signifikante Hemmwirkung auf die Parasiten beobachtet. Die Parasit?mie blieb bei 1. 00 %.

Diese Arbeit wurde durch die Stiftung von “National Nature Science Foundation of China” finanziert (No. 39260066)     

Genetic and Environmental Causes of Tracking in Explosive Strength during Adolescence

Purpose: To determine whether the observed phenotypic stability in explosive strength during adolescence, as measured by inter-age correlations in vertical jump (VTJ), is mainly caused by genetic and/or environmental factors. Methods: Subjects are from the Leuven Longitudinal Twin Study (LLTS) (n = 105 pairs, equally divided over five zygosity groups). VTJ data were aligned on age at peak height velocity (APHV) to attenuate the temporal fluctuations in inter-age correlations caused by differences in timing of the adolescent growth spurt. Simplex models were fitted using structural equation modelling. Results: After aligning the data on APHV, the annual inter-age correlations show a clear simplex structure over a 4 year interval. The best fitting models included additive genetic and unique environmental sources of variation. Heritability estimates ranged between 60.8% (CI 37.7%–77.2%) and 87.3% (CI 74.2%–94.0%) for boys and between 76.5% (CI 56.7%–89.0%) and 88.6% (CI 77.8%–94.1%) for girls. Up to 56.4% and 62.8% of the total variation at the last measurement occasion is explained by additive genetic factors that already explained a significant amount of variation at previous measurement occasions in boys and girls respectively. It thus can be concluded that the observed stability of explosive strength during adolescence is mainly caused by a stable genetic influence in boys and girls. Conclusions: Additive genetic factors seem to be the main cause of the observed phenotypic stability in VTJ performance in boys and girls during adolescence.     

A natriuresis receptor at the carotid bifurcation specifically activated by oxytocin

Summary Rats anaesthetised with Inactin, body temp. maintained at 37°C, were infused with mannitol-saline until both urine flow rate and conductivity reached a balanced state. In separate experiments under analogous conditions cardiac output was measured by dye dilution and organ flow rates by⁸⁶Rb distribution. Doses of oxytocin of 3 ng or less, injected at or just below the carotid bifurcation, caused a highly significant natriuresis with increased tubular rejection, but no measureable haemodynamic changes. The same oxytocin dose given into the internal or external carotid artery above the bifurcation caused neither haemodynamic changes nor natriuresis. Injection of vasopressin, angiotensin and -MSH at the sensitive site did not result in natriuresis in the same dosage range. Section of the sinus nerve significantly decreased the natriuretic response to oxytocin. It is suggested that the carotid body contains a specific oxytocin receptor capable of eliciting natriuresis in the rat.     

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.     

Plasmareninaktivität nach Orthostase und nach Diazoxid bei primärer und renaler Hypertonie

Zusammenfassung 1. Nach Blutdrucksenkung durch schnelle intravenöse Injektion vonDiazoxid kommt es bei 35% der Kranken mit primärem Hochdruck zu einem Anstieg der Plasmareninaktivität. Das Ausbleiben des Reninanstiegs bei der Mehrzahl der fortgeschrittenen primären Hypertonie wird, ebenso wie die abgeschwächte Stimulierbarkeit der Reninsekretion durch Natriummangel, auf eine (adaptiv) verminderte Empfindlichkeit der intrarenalen Receptoren zurückgeführt. Die Blutdrucksenkung durch Diazoxid ist bei positiv und negativ reagierenden Kranken gleich groß.2. Nach einer definiertenOrthostase von 60 min Dauer bei 70° Schräglagerung fehlt ein Plasmareninanstieg bei 63% der untersuchten Patienten mit primärer Hypertonie, wofür ebenfalls eine herabgesetzte Ansprechbarkeit intrarenaler Receptoren verantwortlich gemacht werden muß. Eine Orthostase-negative Reaktion wird ebenso wie eine solche nach Diazoxid häufiger bei primärer Hypertonie in einem fortgeschrittenem, aber auch schon in einem frühen Stadium beobachtet, in dem der Plasmareninanstieg auf Natriumentzug noch intakt ist.3. Die Reninsekretion verhält sich beiprimärer Hypertonie nach Diazoxid, Orthostase und Natriumentzug nicht übereinstimmend, was für die Existenz verschiedener intrarenaler Mechanismen spricht, die die Reninfreisetzung im iuxtaglomerulären Apparat beeinflussen. Patinten mitrenalem Hochdruck weisen dagegen ein übereinstimmendes positives oder negatives Verhalten der Reninsekretion nach Diazoxid und nach Orthostase auf.4. Für diedifferentialdiagnostische Abgrenzung einer funktionell wirksamen Nierenarterienstenose von einer primären Hypertonie ist weder der Diazoxidtest noch — ohne gleichzeitigen Natriumentzug — in der durchgeführten Versuchsanordnung der Orthostasetest geeignet. Zum Nachweis einer funktionell wirksamen Nierenarterienstenose empfiehlt sich die getrennte Plasmareninbestimmung in den Nierenvenen oder als Suchmethode die Doppelstimulation durch 3tägigen Natriumentzug und Orthostase am letzten Tag der Natriumrestriktion.

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Summary 1. After blood pressure reduction by rapid intravenous injection of diazoxide plasma renin activity is increased in 35% of primary hypertension. The absence of plasma renin increase in the majority of advanced primary hypertension is explained by an (adaptive) diminished sensibility of the intrarenal receptors. The reduction of blood pressure is equal in diazoxide responsive and unresponsive patients.2. A plasma renin increase failed in 63% of primary hypertension after upright posture by tilting to 70° for 60 minutes. This result is also explained by a diminished sensibility of the intrarenal receptors for renin secretion. An unresponsive reaction to upright posture and diazoxide is more frequent in advanced primary hypertension, but is observed also in an early stage, in which plasma renin stimulation by sodium deficiency is maintained.3. The stimulation of the renin secretion in primary hypertension is not equal after upright posture, diazoxide and sodium restriction supporting the existence of several intrarenal mechanism controlling the iuxtaglomerular apparatus. In renal hypertension on the contrary, there is a corresponding positive or negative reaction of renin secretion after diazoxide and upright posture.4. For the differential diagnosis of a functional renal artery stenosis neither the diazoxide test nor — without sodium restriction — the orthostase test is suitable. For the evidence of the functional significance of a renal artery stenosis the separated estimation of plasma renin activity in both renal veins or as a sreening test the double stimulation by sodium restriction for 3 days and upright posture is recommended.

     

Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.

uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface. As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods. Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen. The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized. All MAbs reacted under reducing conditions in immunoblot analyses with E. coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells. Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well. By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best. Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR). In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors. Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA. As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7 and IIIB11 may be useful for immunohistochemical studies of uPAR expression in tissue remodeling processes in tumor invasion. In conclusion, we have devised well defined and epitope-mapped MAbs to uPAR that are highly specific tools for detection and targeting of uPAR in tumor tissue.     

Disruption of mCry2 restores circadian rhythmicity in mPer2 mutant mice

Many biochemical, physiological, and behavioral processes display daily rhythms generated by an internal timekeeping mechanism referred to as the circadian clock. The core oscillator driving this clock is located in the ventral part of the hypothalamus, the so called suprachiasmatic nuclei (SCN). At the molecular level, this oscillator is thought to be composed of interlocking autoregulatory feedback loops involving a set of clock genes. Among the components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) and Cryptochrome 1 and 2 (mCry1 and mCry2) genes. A mutation in the mPer2 gene leads to a gradual loss of circadian rhythmicity in mice kept in constant darkness (DD). Here we show that inactivation of the mCry2 gene in mPer2 mutant mice restores circadian rhythmicity and normal clock gene expression patterns. Thus, mCry2 can act as a nonallelic suppressor of mPer2, which points to direct or indirect interactions of PER2 and CRY2 proteins. In marked contrast, inactivation of mCry1 in mPer2 mutant mice does not restore circadian rhythmicity but instead results in complete behavioral arrhythmicity in DD, indicating different effects of mCry1 and mCry2 in the clock mechanism