Influence of 19-nortestosterone and androgens on progesterone biosynthetic enzymes in early human placental explants

We recently showed that the production of progesterone (P4) in human placental explant culture from early gestation is enhanced by treatment with 19-nortestosterone (19-NT) or with certain androgens, namely androsen, namely androstenedione (A-dione), 5-androstane-3, 17β diol (3-diol) and 5-androstane-3β, 17β diol (3β-diol). This stimulation of P4 was explored further in this study. There was little metabolism of radioactive P4 when incubated for 24 h in the presence or absene of these steroids. The role of different steroids in the regulation of P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was evaluated by measuring the conversion of P4 derived from unlabelled 25-hydroxycholesterol and from labelled pregnenolone, respectively. The results showed that 19-NT, A-dione and 3-diol stimulated (P450scc) activity; however, 3β-diol was ineffective. While 19-NT and 3β-diol enhanced the bioconversion of pregnenoloe to P4, A-dione and 3-diol were without effect.

The initial rapid stimulation of P4 by 19-NT within 2 h of incubation was not blocked by concurrent treatment with cycloheximide (CH). However, after incubation for 24 h, 70% of the 19-NT-stimulated P4 was abolished by CH. During the same incubation period,] P4 stimulation by A-dione, 3- and 3β-diol were completely blocked by treatment with CH. Thus our observations suggest that 19-NT-stimulated P4 accumulation is due to the combined effects on P450scc adn 3β-HSD enzyme activities. A-dioneand 3-diol increase biosynthesis of P4 by acting selectively on P450scc enzyme. However, the stimulatory action of 3β-diol on P4 is only at the level of 3β-HSD. Since CH blocks the stimulatory actions, the mechanism(s) by which androgens (A-dione, 3-diol and 3β-diol) and norandrogen (19-NT) augment the biosynthetic enzyme activities appears to be mediated by a process inhibited by CH. Since CH interference was absent during the initial rapid P4-stimulation by 19-NT, there may be a direct action of this steroid at the cellular level which is not dependent on new protein synthesis.     

An evaluation of educators’ views on the e-Bug resources in England

Introduction: e-Bug is an international educational resource for young people covering microbes, hygiene and antibiotics. e-Bug supports NICE guidance on changing public behaviour around antibiotic use. This study aimed to determine educators’ views of the e-Bug teacher resources to inform further development and dissemination of e-Bug. Methods: Age appropriate e-Bug resource packs were posted to every primary school (N = 19,142) and secondary school (N = 5637) in England with a cover letter signed by the Chief Medical Officer, Chief Executive of PHE and e-Bug Project Lead inviting educators to complete an online survey to evaluate the e-Bug resources. The online survey consisted of nine questions and took approximately 15 min to complete. Results: 695 participants completed the online survey. 94% of participants rated the e-Bug resource as excellent or good; one fifth of respondents used the e-Bug resources at least termly. Educators who used e-Bug rated the different lesson plans as excellent or good including ‘Introduction to Microbes’ (98%) and ‘Hand Hygiene’ (95%). Educators provided suggestions for the development of additional lessons plans. Conclusions: Educators view e-Bug as a valuable resource for teaching children about hygiene and antibiotics. Further e-Bug promotion and resource development is required to increase awareness and usage in schools.     

Airborne fungal spores in 80 homes in the Latrobe Valley, Australia: levels, seasonality and indoor-outdoor relationship

Airborne viable and total fungal spores were sampled inside and outside 80 houses in the Latrobe Valley, Victoria, Australia as part of a larger indoor environmental study. Each residence was visited six times over a period of 1 year for sample collection, and fungal spore samples were collected from at least three indoor sites and from an outdoor site. Viable spores were sampled using an Andersen sampler, while total spores were assessed using a Burkard spore trap. Identification of fungal colonies to genera level was performed in two seasons; winter and late spring. The most common fungal genera/groups wereCladosporium, Penicillium, and yeasts, both indoors and outdoors in winter and late spring. Outdoor levels were higher than those indoors throughout the year, and a significant seasonal variation in spore levels was seen both indoors and outdoors with overall maxima in summer. Contrary to this trend, the levels ofAspergillus, yeasts,Cephalosporium andGliocladium were higher in winter. Most fungal genera were found in greater concentrations outdoors compared to indoors, butPenicillium was more common indoors. Outdoor spore levels were a significant influence on indoor levels, but seasonal differences suggest that other influences are important.     

Leishmaniavirus-Dependent Metastatic Leishmaniasis Is Prevented by Blocking IL-17A

Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5–10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients.     

trans-4-hydroxy-β-prolinebetaine,a new betaine fromFurcellaria lumbricalis

From the red marine alga Furcellaria lumbricalis (Huds.) Lamour, a novel betaine has been isolated and characterised from infra-red and nuclear magnetic resonance spectroscopic and mass spectrometric data astrans-4-hydroxy-β-prolinebetaine.     

Amplification of a new region of DNA in an unselected laboratory stock of L. tarentolae: the T region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.     

Histochemical demonstration of 5-hydroxytryptamine in poison glands of amphibian skin

Summary The aim of this project was to see whether 5-hydroxytryptamine (5-HT) believed to be present in the skin glands of anuran amphibians can be demonstrated histochemically. The Periodic acid-Schiff technique (P.A.S.) and three histochemical methods known to demonstrate 5-HT in enterochromaffin cells (Pontana's, diazonium and Schmorl's) were applied to the dorsal skin of three species—Xenopus laevis, Rana angolensis and Bufo regularis.Mucous glands were identified by their P.A.S. reactivity in all three species. Poison glands were identifiable in Bufo regularis and Xenopus laevis only. The secretory granules of the latter glands have strong positive reactions with all three histochemical techniques used.It is concluded that the poison glands of anuran amphibians contain 5-HT demonstrable by histochemical means.     

A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.     

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl₂ was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.Abbreviations Chl chlorophyll - DCPIP dichlorophenolindophenol - DPC diphenolcarbazide - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane