Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

Investigation of arsenic-stressed yeast (Saccharomyces cerevisiae) as a bioassay in homeopathic basic research.

This study investigated the response of arsenic-stressed yeast (Saccharomyces cerevisiae) towards homeopathically potentized Arsenicum album, a duckweed nosode, and gibberellic acid. The three test substances were applied in five potency levels (17x, 18x, 24x, 28x, 30x) and compared to controls (unsuccussed and succussed water) with respect to influencing specific growth parameters. Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. Yeast grew in microplates over a period of 38 h in either potentized substances or water controls with 250 mg/l arsenic(V) added over the entire cultivation period. Yeast's growth kinetics (slope, Et50, and yield) were measured photometrically. The test system exhibited a low coefficient of variation (slope 1.2%, Et50 0.3%, yield 2.7%). Succussed water did not induce any significant differences compared to unsuccussed water. Data from the control and treatment groups were both pooled to increase statistical power. In this study with yeast, no significant effects were found for any outcome parameter or any homeopathic treatment. Since in parallel experiments arsenic-stressed duckweed showed highly significant effects after application of potentized Arsenicum album and duckweed nosode preparations from the same batch as used in the present study, some specific properties of this experimental setup with yeast must be responsible for the lacking response.

Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate.

OBJECTIVE: This study examines if homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can elicit ameliorative responses in yeast (Saccharomyces cerevisiae) exposed to arsenate. METHODS: The yeast S. cerevisiae 699 was cultured in a standard yeast extract peptone dextrose broth medium. It was exposed to the final concentration of 0.15 mmol/L arsenate for two intervals, 1 h and 2 h, respectively. The cell viability was determined along with the assessment of several toxicity biomarkers such as catalase (CAT), superoxide dismutase (SOD), total thiol (GSH) and glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation, protein carbonylation and DNA damage. Reactive oxygen species (ROS) accumulation, expressions of relevant stress transcription activators like Yap-1 and Msn 2, and mRNA expression of yeast caspase-1 (Yca-1) were also measured. RESULTS: Treatment of arsenate increased lipid peroxidation, protein carbonylation, DNA damage, ROS accumulation and expressions of Yap-1, Msn 2 and Yca-1 and decreased GSH, G6PDH, CAT and SOD. Ars Alb 30C administration decreased lipid peroxidation, protein carbonylation, DNA damage, ROS formation and Msn 2 and Yca-1 expressions and increased cell viability, GSH, G6PDH, CAT and SOD significantly (P<0.05), except for a slight increase in Yap-1 expression. CONCLUSION: Ars Alb 30C triggers ameliorative responses in S. cerevisiae exposed to arsenate.

Evaluation of antipyretic activity of Belladonna and Pyrogenium ultrahigh dilutions in induced fever model.

Background Belladonna and Pyrogenium are commonly used to treat fever in homeopathy. But in vivo antipyretic activity of these medicines is not reported yet. The study was conducted to evaluate the effectiveness of ultrahigh dilutions of Belladonna (Bell) and Pyrogenium (Pyro) in fever model of rabbits induced by Baker's yeast. Methods Healthy, local strain rabbits (♂ and ♀) were divided into seven groups (n=42): Normal control, negative control, standard control, pyro 1000c, pyro 200c, Bell 1000c and Bell 200c. Fever was induced by intra peritoneal injection of 135 mg/kg Baker's yeast suspension. Rectal temperature was measured hourly. All the medicines were administered once a day. The results were expressed as mean ± SEM. ANOVA and least significant difference post hoc test were applied for checking the level of significance, p-value of ≤0.05 was considered significant statistically. Results Pyro in both potencies significantly reduced fever in rabbits compared to negative control group, while both potencies of Bell were ineffective. Paracetamol and Pyro 1000c reduced by 1.2 °C (39.7 ± 0.1 to 38.5 ± 0.1), while Pyro 200c reduced by 1 °C temperature (39.7 ± 0.5 to 38.7 ± 0.2). Conclusions Pyro possesses marked antipyretic activity in rabbit's Baker's yeast fever model. It would embolden its clinical use in fever with more guarantee of its efficacy. However, caveat of small sample size necessitates replication of experiment in large sample size.

Effects of homoeopathic ultrahigh dilutions of Aconitum napellus on Baker's yeast-induced fever in rabbits.

OBJECTIVE: To evaluate the effects of homoeopathic ultrahigh dilutions of Aconitum napellus in Baker's yeast-induced fever in rabbits. METHODS: Rabbits were divided into 4 groups and each group contained 6 rabbits. Baker's yeast suspension (20%) was injected subcutaneously. After fever induction, paracetamol and homoeopathic ultrahigh dilutions (A. napellus 200c and 1 000c) were given orally. Rectal temperature was measured with digital thermometer hourly. RESULTS: Fever was induced in all the rabbits after 4 hours of Baker's yeast administration. A. napellus 200c and 1000c significantly reduced the temperature (P < 0.05). In positive control, temperature decrease was more significant (P < 0.001). CONCLUSION: The above findings indicate the effectiveness of ultrahigh dilutions of A. napellus in Baker's yeast-induced fever in rabbits. However, the effects were slower and less significant than standard medicine. Moreover, future research is required to know their mechanism of reducing temperature.

Homeopathy on cultures of Saccharomyces cerevisiaeand impact on fermentation

Studies have shownthat homeopathy modulates the activity of both single-and multi-celled organisms;therefore, we propose a study into the action of Arnica Montanaand S. cerevisiae fungus nosode on growth "in vitro", and on the fermentation of S. cerevisiaeon brewer's wort. Methods:250 µL of medication in 30% alcohol were placed in 5 mL of Sabouraud Broth (SB) or wort, with 20 µL of fungus ata McFarland standard of 0.5 and in a dilution of 1:100. Fungal growth was evaluated via spectrophotometry at 600 nm or a cell count in a Neubauer chamber in a kinetic of 1 to 5 days' incubation at 25ºC. The production of alcohol by the fungus was evaluated using the BRIX index in the samekinetic. 1x107fungi/mL were previously incubated with medication for 5 days and, afterwards, placed in 20 mL of fresh wort, incubated at 25ºC for 7 days and evaluated for growth and sugar consumption. Resultsand Discussion: The SB results revealed that after 2days incubation with Arnica30CH, an increase in fungal growth was observed (p<0.0001), whilewith nosode 6 and 30CH there was a reduction in growth after 2 and 5 days incubation (p<0.001). The fungi incubated with Arnica30CH exhibited increased sugar consumption after 2 and5 days incubation (p<0.05), while the nosode 30CH resulted in lower sugar consumption after 2 and 3 days incubation (p<0.05). The results for fungal growth and sugar consumption with the wort were similar to those using SB.The fungalcultures previously incubated with homeopathic medication and subsequent incubation with fresh wortindicated a loss of distinction, bothin terms of fungal growth and sugar consumption. This piece of data may suggest action by the homeopathic medication only when in contact with the cells. Conclusion: The treatment of the S. cerevisiae fungus using Arnica and the S. cerevisiae nosode produced a significant modulation in fungal growth and sugar consumption.

Phagocytosis, endosomal/lysosomal system and other cellularaspects of macrophage activation by Canova medication.

Canova is a homeopathic medication with immunomodulatory properties, recommended for diseases where the immune system is depressed. Our research aims to study the activation of mice peritoneal macrophages when submitted to in vivo and in vitro Canova treatment. Morphological parameters and acid phosphatase activity were analyzed using light and transmission electron microscopy. Differential interference contrast microscopy, including serial time acquisition in living cells, was also performed. The results demonstrated a greater spreading ability in Canova treated macrophages, a higher phagocytic activity of non-infective microorganisms (Saccharomyces cerevisiae and Tripanosoma cruzi epimastigotes) and a tendency to lower the phagocytic activity of the infective microorganisms T. cruzi trypomastigotes and Leishmania amazonensis, when compared with control cells. Acid phosphatase activity was analyzed and showed that Canova treatment stimulates an increase of the endosomal/lysosomal system. Treated macrophages that do or do not interact with yeast present a higher number of acid phosphatase marked vesicles compared to control cells. In contrast, the activity of tartrate resistant acid phosphatase (TRAP), is lower in Canova treated macrophages. The net results demonstrate that Canova medication is an effective stimulator of macrophage activity.

The origin of hormesis: historical background and driving forces.

Copious historical reviews of Calabrese and Baldwin (Hum Exp Toxicol 2000; 19: 2-31; 32-40) attribute the description of the reversal of cellular activities from stimulation at low doses to inhibition at high doses by Schulz (Pflüg Arch 1988; 42: 517-41) as the prioritizing contribution to the phenomenon which was later called hormesis. However, an extended search of the older literature uncovers Virchow (Virch Arch 1854; 6: 133-34) as the first descriptor, three and a half decades in advance of Schulz. Virchow observed an increase of the beating activity of the ciliae of tracheal epithelia of postmortem mucosa by sodium and potassium hydroxide at low concentrations, and a concentration-dependent decrease to arrest at higher concentrations. This observation constituted a cornerstone in Virchow's 'cellular pathology', which was based on the theory of cellular 'irritation and irritability'. Schulz's experiment was essentially triggered by the psychiatrist Rudolf Arndt, an ardent protagonist of homeopathy. Schulz's pre-occupation with homeopathic principles, which dominates his scientific oeuvre over his lifetime, may be seen as one of the reasons for the marginalization of hormesis.

Application of membrane and ozonation technologies to remove color from agro-industry effluents.

The results of membrane and ozonation experiments carried out on various agro-industry effluents including fermentation (baker's yeast), corrugated board, opium alkaloid and textile dying industries are presented. The experiments were performed using lab-scale membrane and ozonation reactors. Color removals were in the range of 80 to 99% for the membrane treatment studies. Ozonation experiments have shown that color removals in the range of 83 to 98% are possible for the investigated wastewaters. Final color levels were lower than 100 Pt-Co unit, which is quite acceptable aesthetically. The relative unit treatment costs of ozonation were about two times higher than membrane systems especially for very strong colored effluents including fermentation and opium alkaloid industries. The study has demonstrated that both membrane and ozonation technologies are viable options for color removal.

Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

BACKGROUND: Homeopathic potencies are used as specific remedies in complementary medicine. Since the mode of action is unknown, the presumed specificity is discussed controversially. OBJECTIVE: This study investigated the effects of potentised substances on two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, in a stable and reliable test system with systematic negative controls. MATERIALS AND METHODS: Yeast cells were cultivated in either potentised substances or water controls in microplates and their growth kinetics were measured photometrically. Water control runs were performed repeatedly to investigate the stability of the experimental set-up (systematic negative controls). RESULTS: 4 out of 14 screened substances seem to have affected the growth curve parameters slope or yield. Out of these substances, azoxystrobin and phosphorus were chosen for 8 further replication experiments, which partly confirmed the results of the screening. On the average of all experiments, azoxystrobin affected the slope of the growth curve of Saccharomyces cerevisiae (p < 0.05), and phosphorus affected the slope of the growth curve of Schizosaccharomyces pombe (p < 0.05). No effects were seen in the water control runs. In addition, significant interactions between treatment with potentised substances and experiment number were observed in all experiments with potentised substances (p < 0.01), but not in the water control runs. CONCLUSIONS: Both yeast species reacted to certain potentised substances by changing their growth kinetics. However, the interactions found point to additional factors of still unknown nature, that modulate the effects of potentised substances. This stable test system with yeasts may be suitable for further studies regarding the efficacy of homeopathic potencies.

Efectos de los campos magnéticos sobre el crecimiento de saccharomyces cerevisiae / Effects of magnetics fields on saccharomyces cerevisiae growth

Cultivos de saccharomyces cerevisiae fueron sometidos al efecto de campos magnéticos variables de alta frecuencia (hasta 100KHz) y bajas densidades de flujo (hasta 620mG). Se aplicó la metodología estadística de superficies de respuesta para evaluar en qué rangos de las variables densidad de campo, frecuencia y tiempo de aplicación, se obtenían efectos significativos sobre la concentración celular (CB). En los valores de las variables predichos como significativos por los modelos obtenidos de las superficies de respuesta, se hicieron comparaciones entre el crecimiento del cultivo a diferentes densidades de flujo, tiempos de exposición al campo y frecuencias, para evaluar el comportamiento de la CB ante la modificación de una sola variable a la vez. Se utilizó el metodo de la mínima diferencia significativa (LSD) para comparar los resultados de los ensayos correspondientes a cada variable. Los resultados mostraron que se puede estimular el crecimiento del cultivo tratado con campos magnéticos, cuando se utilizan campos con densidades de flujo de 20mG, frecuencias de 100kHz y tiempos de aplicación de 30s. Los porcentajes de estimulación obtenidos con este tratamiento fueron del 30 por ciento con respecto a un cultivo control

Comment on the paper, "Growth of yeast cultures as in vitro model for investigationg homoeopathic remedies"

A recent paper by Steffen repeating earlier work by Jones et al. failed to confirm any effect of potencies of Pulsatilla on the growth rate of cultures of Saccharomyces cerevisiae which had previously been reported. We find that using a more specific statistical test on the results given by Steffen it can be shown that these results contain almost the same periodicity with potency that Jones et al. reported, although only corresponding to a variation of +or- l.5//about the mean value