Dodecylamine rapidly kills of spores of multiple Firmicute species: properties of the killed spores and the mechanism of the killing.

AIMS: Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. METHODS AND RESULTS: Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. CONCLUSIONS: This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

Killing of bacterial spores by dodecylamine and its effects on spore inner membrane properties.

AIMS: The objective of this study was to determine the effects of Ca-dipicolinic acid (CaDPA), cortex-lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. METHODS AND RESULTS: Bacillus subtilis spores, wild-type, CaDPA-less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine-treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild-type and CaDPA-less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l-1 dodecylamine in 4 h at 45°C with spores at ~108  ml-1 . Dodecylamine killing of decoated wild type and CLE-less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA-less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA-replete spores or dodecylamine-killed CLE-less spores peripherally, and cores of decoated CaDPA-less spores and dodecylamine-killed intact spores with CLEs. The IM of some decoated CaDPA-less spores was greatly reorganized. CONCLUSIONS: Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. SIGNIFICANCE AND IMPACT OF THE STUDY: This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained.

Lack of efficient killing of purified dormant spores of Bacillales and Clostridiales species by glycerol monolaurate in a non-aqueous gel.

Inactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20 h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5 h but gave ~1·5 log killing at 24 h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5 h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores.

Juara Sihat: assessing the sustained impact of a school-based obesity intervention.

INTRODUCTION: Obesity and physical inactivity among school children are among the most challenging health problems in Malaysia. The present study aimed to evaluate the sustained impact of Juara Sihat programme on physical activity level and anthropometric status at 18-month post-intervention. METHOD: Participants of Juara Sihat (n=55) were followed-up at 18 months after completion of the intervention. Juara Sihat intervention was implemented over 12 weeks and focused on four key components: (i) five one-hour nutrition education classes, (ii) four one-hour physical activity education sessions, (iii) family involvement, and (iv) empowerment of Parents and Teachers Association. Anthropometric variables (body mass index, body fat percentage and waist circumference) were measured and physical activity level was evaluated by using Physical Activity Questionnaire for Children (PAQ-C) at baseline (P0), immediately upon completion of intervention (P1), at three-month post-intervention (P2), and at 18-month postintervention (P3). Analyses of repeated measures analysis of covariance (ANCOVA) with intention-to-treat principle were applied. RESULTS: Sustained effects were found in BMI-for-age z-score which showed a reduction (P0 2.41±0.84 vs P3 2.27±0.81) and physical activity level which showed positive improvements (P0 2.46±0.62 vs P3 2.87±0.76) at 18 months after intervention was completed. Body fat and waist circumference had increased over the same time period. CONCLUSION: Overall, this study successfully demonstrated sustained intervention effects of Juara Sihat intervention on BMI-for-age z-score and physical activity, but not on body fat percentage and waist circumference.

Automated computer-based CT stratification as a predictor of outcome in hypersensitivity pneumonitis.

BACKGROUND: Hypersensitivity pneumonitis (HP) has a variable clinical course. Modelling of quantitative CALIPER-derived CT data can identify distinct disease phenotypes. Mortality prediction using CALIPER analysis was compared to the interstitial lung disease gender, age, physiology (ILD-GAP) outcome model. METHODS: CALIPER CT analysis of parenchymal patterns in 98 consecutive HP patients was compared to visual CT scoring by two radiologists. Functional indices including forced vital capacity (FVC) and diffusion capacity for carbon monoxide (DLco) in univariate and multivariate Cox mortality models. Automated stratification of CALIPER scores was evaluated against outcome models. RESULTS: Univariate predictors of mortality included visual and CALIPER CT fibrotic patterns, and all functional indices. Multivariate analyses identified only two independent predictors of mortality: CALIPER reticular pattern (p = 0.001) and DLco (p < 0.0001). Automated stratification distinguished three distinct HP groups (log-rank test p < 0.0001). Substitution of automated stratified groups for FVC and DLco in the ILD-GAP model demonstrated no loss of model strength (C-Index = 0.73 for both models). Model strength improved when automated stratified groups were combined with the ILD-GAP model (C-Index = 0.77). CONCLUSIONS: CALIPER-derived variables are the strongest CT predictors of mortality in HP. Automated CT stratification is equivalent to functional indices in the ILD-GAP model for predicting outcome in HP. KEY POINTS: • Computer CT analysis better predicts mortality than visual CT analysis in HP. • Quantitative CT analysis is equivalent to functional indices for prognostication in HP. • Prognostication using the ILD-GAP model improves when combined with quantitative CT analysis.

Contrast-enhanced spectral mammography in recalls from the Dutch breast cancer screening program: validation of results in a large multireader, multicase study.

OBJECTIVES: Contrast-enhanced spectral mammography (CESM) is a promising problem-solving tool in women referred from a breast cancer screening program. We aimed to study the validity of preliminary results of CESM using a larger panel of radiologists with different levels of CESM experience. METHODS: All women referred from the Dutch breast cancer screening program were eligible for CESM. 199 consecutive cases were viewed by ten radiologists. Four had extensive CESM experience, three had no CESM experience but were experienced breast radiologists, and three were residents. All readers provided a BI-RADS score for the low-energy CESM images first, after which the score could be adjusted when viewing the entire CESM exam. BI-RADS 1-3 were considered benign and BI-RADS 4-5 malignant. With this cutoff, we calculated sensitivity, specificity and area under the ROC curve. RESULTS: CESM increased diagnostic accuracy in all readers. The performance for all readers using CESM was: sensitivity 96.9 % (+3.9 %), specificity 69.7 % (+33.8 %) and area under the ROC curve 0.833 (+0.188). CONCLUSION: CESM is superior to conventional mammography, with excellent problem-solving capabilities in women referred from the breast cancer screening program. Previous results were confirmed even in a larger panel of readers with varying CESM experience. KEY POINTS: • CESM is consistently superior to conventional mammography • CESM increases diagnostic accuracy regardless of a reader's experience • CESM is an excellent problem-solving tool in recalls from screening programs.

Functional assessment in the multimodality imaging of pancreatic neuro-endocrine tumours.

Pancreatic neuroendocrine tumours comprise a heterogeneous group that are rare but could result in serious manifestations. Surgical excision provides the best approach to treatment but many patients may have small lesions that are difficult to detect, or present with wide spread disease by the time of diagnosis. In addition to clinical assessment and biochemical tests, imaging is a major factor in establishing the diagnosis. Cross-sectional imaging such as US, CT and MR, play a major role in the initial assessment. However, they may miss small lesions or metastatic spread. Functional Imaging became possible with the development of somatostatin receptor imaging using 111In-octreotide, which when combined with anatomical imaging could provide enhanced detection. A major improvement has been achieved by combining receptor and PET inaging through the use of 68Ga-DOTA complexes that have been shown to have a much better sensitivity than other imaging modalities and can provide the basis for radionuclide treatment with 90Y or 177Lu labelled with DOTA complexes.

Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex.

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

Fruit and Vegetable Consumption among Special School Students with Mild Intellectual Disability in Hong Kong.

OBJECTIVE: The aim of this study was to predict the fruit and vegetable consumption intention of students with mild intellectual disability in Hong Kong by the application of Ajzen's Theory of Planned Behaviour. METHODS: 50 students with mild intellectual disability (30 male and 20 female), ranging in age from 15 to 38 years, were participated in this study. By means of face-to-face interviews, demographic data, Food Preference and variables of Theory of Planned Behaviour, such as Attitude, Subjective Norm and Perceived Behavioural Control were measured. RESULTS: 20%, 28% and 10% students with mild intellectual disability were rated to be overweight, obese and severely obese respectively. The rest of 10% were classified to be underweight. Regarding the daily intake of fruit and vegetable, 96% students with mild intellectual disability failed to consume sufficient amount. The variables of Theory of Planned Behaviour explained 47.7% of fruit and vegetable consumption intention with significant factors of Attitude, Subjective Norm and Perceived Behavioural Control. Food Preference was found to be a useful construct and further improve the prediction by about 7% after incorporating into the model. CONCLUSIONS: Results of this study indicated that Theory of Planned Behaviour is a useful model to predict dietary intention of students with mild intellectual disability in Hong Kong. Food Preference was a significant predictor to model the intention of fruit and vegetable consumption among students other than Attitude, Subjective Norm and Perceived Behavioural Control.

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Decoloration of textile dyes by alginate-immobilized Trametes versicolor.

Alginate-immobilized Trametes versicolor decolorized Amaranth at similar rates in repeated batch culture when the dye was present in either (i) modified Kirk's medium containing 0.22 gl(-1) ammonium tartrate, (ii) the same buffer, thiamine, trace elements and glucose concentrations as in the modified Kirk's medium, or (iii) glucose alone at either 1, 5 or 10 gl(-1). With glucose alone (0.5 gl(-1)), Amaranth, Reactive Black 5, Reactive Blue 19 and Direct Black 22 had first-order decoloration rate constants of 0.56, 0.76, 0.52, and 0.15 h(-1), respectively. Mixtures of these dyes were also completely decolorized. After four successive decolorations, beads were kept in storage solutions for 48 d at 6 degrees C. CaCl2 (1g l(-1)) was the best storage solution as the beads were easier to handle and had the fastest decoloration rates after storage. Decoloration rates were faster with lower viscosity (less than 2000 cps) alginates and with softer beads which had a lower resistance to compression. Fungal colonization of the beads resulted in higher biomass concentrations with a corresponding higher decoloration rate but the beads became larger, had a lower resistance to compression and a higher percentage of bead breakage in a stirred tank reactor. Biomass, recovered from beads in which there was no growth, could be dispersed while the biomass from colonized beads formed a hollow, spherical shell due to growth on and near the bead surface and no growth in the bead interior. If alginate-immobilized T. versicolor is to be used in a stirred tank reactor, a high biomass loading during the immobilization phase and no fungal growth in the beads is recommended to have high decoloration rates and low bead breakage.

Patterns of tooth loss in young adult Hong Kong Chinese patients in 1983 and 1998.

OBJECTIVE: To describe and compare patterns of tooth loss in 2 groups of 21- to 25-year-old Hong Kong Chinese patients examined 15 years apart. MATERIALS AND METHODS: The panoramic radiographs of consecutive young adult patients who attended the primary care department of the Dental School of the University of Hong Kong in 1983 and 1998 were reviewed. RESULTS: The proportions of patients with full dentition were 36.0% in 1983 and 45.1% in 1998. However, when third molars were excluded, the proportions were 44.3% and 62.3%, respectively. The prevalence of missing first molars was 10.5% and 3.2% for the 1983 and 1998 groups, respectively, whereas that for missing third molars was 13.5% and 17.9% and that for missing premolars was 1.9% and 2.4%, respectively; all of the differences were statistically significant (p < 0.01). Although the first molars, especially the lower first molars, were at greatest risk of being lost in both groups, the prevalence of missing first molars fell substantially (10.5% in 1983, 3.2% in 1998); in contrast, the prevalence of missing premolars and third molars increased. CONCLUSIONS: The decline in the prevalence of missing first molars may in part reflect the efficacy of toothbrushing, whereas the increase in missing premolars and third molars reflects increases in orthodontic and oral surgical activity in the intervening period.

Stage T3a renal cell carcinoma: staging accuracy of CT for sinus fat, perinephric fat or renal vein invasion.

OBJECTIVE: To study the accuracy of CT for staging T3a (TNM 2009) renal cell carcinoma (RCC). METHODS: Unenhanced and nephrographic phase CT studies of 117 patients (male:female = 82:35; age range, 21-86 years) with T1-T3a RCC were independently reviewed by 2 readers. The presence of sinus or perinephric fat, or renal vein invasion and tumour characteristics were noted. RESULTS: Median (range) tumour size was 5.5 (0.9-19.0) cm; and 46 (39%), 16 (14%) and 55 (47%) tumours were pT1, pT2 and pT3a RCC, respectively. The sensitivity/specificity for sinus fat, perinephric fat and renal vein invasion were 71/79%, 83/76% and 59/93% (Reader 1) and 88/71%, 68/72% and 69/91% (Reader 2) with κ = 0.41, 0.43 and 0.61, respectively. Sinus fat invasion was seen in 47/55 (85%) cases with T3a RCC vs 16/55 (29%) and 33/55 (60%) for perinephric fat and renal vein invasion. Tumour necrosis, irregularity of tumour edge and direct tumour contact with perirenal fascia or sinus fat increased the odds of local invasion [odds ratio (OR), 2.5-3.7; p < 0.05; κ = 0.42-0.61]. Stage T3a tumours were centrally located (OR, 3.9; p = 0.0009). CONCLUSION: Stage T3a RCC was identified with a sensitivity of 59-88% and specificity of 71-93% (κ = 0.41-0.61). Sinus fat invasion was the most common invasive feature. ADVANCES IN KNOWLEDGE: Centrally situated renal tumours with an irregular tumour edge, inseparable from sinus structures or the perirenal fascia and CT features of tumour necrosis should alert the reader to the possibility of Stage T3a RCC (OR, 2.5-3.9).

Selective absorption of ultraviolet laser energy by human atherosclerotic plaque treated with tetracycline.

Tetracycline is an antibiotic that absorbs ultraviolet light at 355 nm and preferentially binds to atherosclerotic plaque both in vitro and in vivo. Tetracycline-treated human cadaveric aorta was compared with untreated aorta using several techniques: absorptive spectrophotometry, which demonstrated a distinct absorptive peak at 355 nm in tetracycline-treated plaque that was absent in treated normal vessel; ultraviolet microscopy, which showed that treated atheroma acquired the characteristic fluorescence of tetracycline under ultraviolet light; and tissue uptake of radiolabeled tetracycline, which showed 4-fold greater uptake by atheroma than by normal vessel. In addition, intravenous tetracycline administered to patients undergoing vascular surgery demonstrated characteristic fluorescence in surgically excised diseased arteries. Because of tetracycline's unique properties, we exposed tetracycline-treated and untreated aorta to ultraviolet laser radiation at a wavelength of 355 nm. We found enhanced ablation of tetracycline-treated atheroma compared with untreated atheroma. The plaque ablation caused by ultraviolet laser radiation was twice as extensive in tetracycline-treated vs nontreated plaque (2.2 +/- 0.25 mm vs 1.3 +/- 0.55 mm, p less than 0.017). This study demonstrates the potential of tetracycline plaque enhancement for the selective destruction of atheroma by ultraviolet laser radiation.

Paracoccidioidin and histoplasmin sensitivity in Coari (state of Amazonas), Brazil.

A skin test survey with paracoccidioidin and histoplasmin was conducted on 495 residents of Coari in the Brazilian state of Amazonas. Positive dermal reactions to paracoccidioidin and histoplasmin were seen, respectively, in 13.9% and 50.1% of the study subjects. Specific reaction to paracoccidioidin was observed in 1.6% of the individuals, and to histoplasmin in 37.8%. Cross-reactivity between the two antigens in persons who reacted positively to both skin tests was not totally reciprocal. There were no significant differences in dermal reactivity to either of the two antigens in either sex, or among different occupational groups. No precipitating antibody to paracoccidioidin or histoplasmin was detected in the sera of the skin test-positive individuals by the immunodiffusion test. Skin test positivity in the general population of Coari reaffirmed the concept of benign, self-limited infection in paracoccidioidomycosis and histoplasmosis. It also identified the equatorial forests of the Upper Amazon Basin as endemic zones of these two mycoses.

Occurrence and experimental infection of toads (Bufo marinus and B. granulosus) with Mycobacterium chelonei subsp. abscessus.

In a survey of 234 Amazonian toads and frogs, six strains of Mycobacterium chelonei subsp. abscessus were isolated from the liver or spleen of four of 66 Bufo marinus (6.1%) and from the kidney or peritoneal fluid of two of 86 B. granulosus (2.3%). There were no histopathological lesions in the viscera of the infected animals. Experimental infection of 29 captive B. marinus and B. granulosus, by the intraperitoneal route, with a pooled inoculum of M. chelonei subsp. abscessus caused five deaths near the end of a 2-month observation period. M. chelonei subsp. abscessus was isolated from the liver, spleen, kidney, gonad, heart and lung of toads killed at various intervals after inoculation, and intracellular acid-fast bacilli were seen in these organs. Histological evidence of invasion of tissues by mycobacteria became apparent from the 45th day after infection. The susceptibility to infection of B. marinus and B. granulosus suggests that these toads may serve as a fortuitous animal host for M. chelonei subsp. abscessus.

Evidence that 5 alpha-pregnan-3 alpha-ol-20-one is the metabolite responsible for progesterone anesthesia.

Tadpoles (Rana catesbeiana) were anesthetized with [3H]-progesterone (PG) in order to identify the steroid responsible for anesthesia. PG and its metabolites in brain were resolved (HPLC) and quantitated following sacrifice at different behavioral endpoints. The results support the hypothesis that 5 alpha-pregnan-3 alpha-ol-20-one mediates the loss and the return of the righting response. The observed levels for PG were not consistent with such a role. Neither PG nor 5 alpha-pregnan-3 alpha-ol-20-one produced analgesia.

Forskolin inhibits platelet-activating factor binding to platelet receptors independently of adenylyl cyclase activation.

The effect of forskolin on platelet-activating factor (PAF) receptor was investigated. Rabbit platelets treated with forskolin showed approximately a 9-fold increase in cAMP levels over the control. After treatment of platelets with forskolin prior to PAF binding, a 30-40% (P < 0.005) decrease in PAF binding was observed. The decrease in PAF binding caused by forskolin was concomitant with a decrease in the physiological responses of platelets induced by PAF. However, this forskolin-induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the action of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAF binding by forskolin and dideoxyforskolin was also observed with isolated platelet membranes. To understand the mechanism of forskolin induced changes in PAF binding, the involvement of a G-protein in this process was investigated. Cells treated with GTP gamma S showed approximately a 25% reduction in PAF binding. Addition of forskolin to the GTP gamma S treated cells resulted in a further reduction in PAF binding, suggesting the action of forskolin was independent of G-protein activation. The data suggests that the action of forskolin was independent of adenylyl cyclase or G-protein involvement. It is speculated that the action of forskolin on PAF binding was due to a direct effect of this molecule and its analog on the PAF receptor itself or to components of the post-receptor signalling for PAF.

Evidence that 3 alpha-hydroxy-5 alpha-pregnan-20-one is the metabolite responsible for anesthesia induced by 5 alpha-pregnanedione in the mouse.

Mice were anesthetized with [3H]5 alpha-pregnanedione (5 alpha). Brain levels for 5 alpha and its metabolites were quantitated and compared at time points following injection and at two behavioral endpoints that are characteristic of the anesthetized state. The results support the hypothesis that 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha), a metabolite of 5 alpha, mediates this anesthetic response, and they weigh against the hypothesis that 5 alpha itself is solely responsible for activity. Anesthesia occurred at 3-8 min following injection. During this period, levels of 3 alpha derived from 5 alpha were comparable to those seen when 3 alpha was administered alone. It is estimated that 5 alpha, if active at all, is at least 3-5 times less potent than 3 alpha.

Synthesis of a [1,2-3H]-labeled pregnanolone.

A method is described for the synthesis and purification of 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. [1,2-3H]progesterone (55 Ci/mmol) was incubated with a homogenate of rat brain tissue. The product was purified by Sephadex chromatography and thin-layer chromatography. The identity and purity of the product were established by successive recrystallizations and high-performance liquid chromatography. A 34% portion of the starting material was converted to 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. The final radiopurity of 3 alpha-hydroxy-5 alpha-pregnan-20-one obtained from four independent preparations was 94% to 99%.