Chemical stabilization of a Δ9-tetrahydrocannabinol prodrug in polymeric matrix systems produced by a hot-melt method: Role of microenvironment pH

This research was conducted in order to fabricate stable polyethylene oxide (PEO)-based transmucosal systems of a Δ⁹-tetrahydrocannabinol (THC) prodrug, a hemisuccinate ester, using a hot-melt method. Since Δ⁹-tetrahydrocannabinol (THC-HS) was heat labile, a series of processing aids were evaluated in order to facilitate hot-melt production at lower temperatures, thereby reducing THC-HS degradation. The stability of THC-HS was influenced both by the processing conditions such as heating time and temperature, and the postprocessing storage conditions. The type of formulation additive also affected the extent of degradation. In the presence of polyethylene glycol (PEG)-400, the percentage of relative degradation of THC-HS to THC was 13.5% and 49.4% at 80°C and 120°C, respectively. In contrast, incorporation of vitamin E succinate (VES) reduced processing degradation to 2.1% and 9.2%, respectively, under the same conditions. Severe degradation of THC-HS was observed during storage, even under freezing conditions (−18°C). A VES-Noveon AA-1 combination was observed to best stabilize the prodrug systems both during processing and postprocessing. Stabilization of THC-HS was achieved in these polyethylene oxide matrices at 4°C, with almost 90% of theoretical drug remaining for up to 8 months. Investigation of the pH effect revealed that the pH of the microenvironment in these polymeric systems could be modulated to significantly improve the stability of THC-HS, degradation being the least in a relatively acidic medium. Published: September 1, 2006     

Preformulation Studies of a Prodrug of Δ9-Tetrahydrocannabinol

Preformulation studies were performed on a hemiglutarate ester prodrug of Δ⁹-tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a T _g close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at −18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO.     

Preparation and Characterization of Inclusion Complexes of a Hemisuccinate Ester Prodrug of Δ<Superscript>9</Superscript>-Tetrahydrocannabinol with Modified Beta-Cyclodextrins

Δ⁹-Tetrahydrocannabinol hemisuccinate (THC-HS), an ester prodrug of Δ⁹-tetrahydrocannabinol (THC) has been investigated for its potential to form inclusion complexes with modified synthetic beta-cyclodextrins (CDs). Phase solubility studies were performed to determine the stoichiometric ratio of complexation of THC-HS with random methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl beta-cyclodextrin (HPBCD). THC-HS/RAMEB and THC-HS/HPBCD solid systems were prepared by lyophilization and the lyophilized complexes were characterized by Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic spectroscopy, and molecular modeling techniques. The formation of inclusion complexes of THC-HS/RAMEB and THC-HS/HPBCD was demonstrated by an A_L type curve with the slopes less than unity by the phase solubility method. The association constants for THC-HS/RAMEB and THC-HS/HPBCD were found to be 562.48 and 238.83 M⁻¹, respectively. The stoichiometry of both of the complexes was found to be 1:1 as determined from the Job's plot. This was confirmed by ¹H NMR and FT-IR techniques. The results obtained from the molecular modeling studies were in accordance with the data obtained from nuclear magnetic resonance and FT-IR. The docking studies revealed the most probable mode of binding of THC-HS with RAMEB in which the alkyl chain was submerged in the hydrophobic pocket of the CD molecule and hydrogen bonding interactions were observed between the hemisuccinate ester side chain of THC-HS and the rim hydroxy groups of RAMEB. The solubility of THC-HS was significantly higher in RAMEB compared to HPBCD. Solid dispersions of THC-HS with CDs will be further utilized to develop oral formulations of THC-HS with enhanced bioavailability.     

Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.     

Investigation of 3 industry-wide applied storage conditions for compound libraries

The appropriate storage conditions for a compound file are a crucial factor for the success of drug discovery projects. In this study, 778 highly diverse compounds dissolved in 100% DMSO were stored under 3 industry-wide accepted storage conditions, and the compound integrity was monitored for a period of 6 months. The storage conditions selected were (1) under argon at +15 degrees C, (2) under argon at -20 degrees C, and (3) under ambient atmosphere at -20 degrees C. Each sample was assessed every 4 weeks by liquid chromatography coupled to mass spectrometry (LC/MS). Based on the resulting experimental data, a statistical projection of compound integrity over a period of 4 years for each of the 3 storage conditions was generated applying a linear mixed-effects model. A moderate loss of compound integrity of 12% was calculated for storage at -20 degrees C under argon, a loss of 21% for storage at -20 degrees C under ambient atmosphere, and a strong decrease of 58% for storage at +15 degrees C under argon over this period. The initial purity of the compounds does also influence the rate of compound degradation. Compounds with an initial purity of 50% to 75% degraded faster than compounds with an initial purity of more than 75%. The results of the study enable the prediction of the point in time, when the purity of a compound population falls below a predefined threshold that would trigger the resolubilization or retirement of the compound population represented by the analyzed samples.     

Kinetics of paclitaxel 2'-N-methylpyridinium mesylate decomposition

This study was designed to examine the kinetics of decomposition of paclitaxel 2'-N-methylpyridinium mesylate (PNMM), a derivative of paclitaxel. Further, the potential for PNMM to act as a prodrug of paclitaxel was assessed in vitro. Stability studies of PNMM were conducted over a pH range of 4.0 to 8.0 at 25 degrees C. The critical micelle concentration (CMC) of PNMM was determined by pulsating bubble surfactometry. Studies of the conversion of PNMM to paclitaxel were conducted in vitro in human plasma. Decomposition of PNMM followed apparent zero-order kinetics. The pH-rate profile exhibited no evidence of acid catalysis down to pH 4.0, while the rate was accelerated under base conditions. Surface tension studies suggested that PNMM formed micelles with a CMC of approximately 34 micro g/mL. Conversion studies in phosphate buffer showed that no more than 5% of PNMM converted to paclitaxel, while in human plasma the conversion was about 25%. The degradation of PNMM was via apparent zero-order kinetics and was dependent upon pH. The observed apparent zero-order kinetics of decomposition of PNMM was consistent with the formation of micelles in phosphate buffer. In buffered aqueous media alone or in human plasma, PNMM did not convert quantitatively to paclitaxel. Thus, the limiting factor in the application of PNMM as a prodrug would appear to be the poor potential to convert to paclitaxel.     

Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions

Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy. Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined. Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions. The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs. This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion. Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months. No effect on the microbial permeability of membrane was observed during storage. The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus. The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.     

Does local heating-induced nitric oxide production attenuate vasoconstrictor responsiveness to lower body negative pressure in human skin?

We tested the hypothesis that local heating-induced nitric oxide (NO) production attenuates cutaneous vasoconstrictor responsiveness. Eleven subjects (6 men, 5 women) had four microdialysis membranes placed in forearm skin. Two membranes were perfused with 10 mM of N(G)-nitro-L-arginine (L-NAME) and two with Ringer solution (control), and all sites were locally heated to 34 degrees C. Subjects then underwent 5 min of 60-mmHg lower body negative pressure (LBNP). Two sites (a control and an L-NAME site) were then heated to 39 degrees C, while the other two sites were heated to 42 degrees C. At the L-NAME sites, skin blood flow was elevated using 0.75-2 mg/ml of adenosine in the perfusate solution (Adn + L-NAME) to a similar level relative to control sites. Subjects then underwent another 5 min of 60-mmHg LBNP. At 34 degrees C, cutaneous vascular conductance (CVC) decreased (Delta) similarly at both control and L-NAME sites during LBNP (Delta7.9 +/- 3.0 and Delta3.4 +/- 0.8% maximum, respectively; P > 0.05). The reduction in CVC to LBNP was also similar between control and Adn + L-NAME sites at 39 degrees C (control Delta11.4 +/- 2.5 vs. Adn + L-NAME Delta7.9 +/- 2.0% maximum; P > 0.05) and 42 degrees C (control Delta1.9 +/- 2.7 vs. Adn + L-NAME Delta 4.2 +/- 2.7% maximum; P > 0.05). However, the decrease in CVC at 42 degrees C, regardless of site, was smaller than at 39 degrees C (P < 0.05). These results do not support the hypothesis that local heating-induced NO production attenuates cutaneous vasoconstrictor responsiveness during high levels of LBNP. However, elevated local temperature, per se, attenuates cutaneous vasoconstrictor responsiveness to LBNP, presumably through non-nitric oxide mechanisms.     

Development and evaluation of a thermoreversible ovule formulation of stampidine,a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5-2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness

In order to find a marker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40 degrees C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4 degrees C to 50 degrees C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.     

Development and validation of a method for the quantitation of Delta9tetrahydrocannabinol in oral fluid by liquid chromatography electrospray-mass-spectrometry

Analysis of Delta(9)tetrahydrocannabinol (Delta(9)THC) and its metabolites in biological samples is of great relevance for forensic purposes. In the case of oral fluid (OF), the analysis should determine Delta(9)THC, whereas in urine, it detects the inactive metabolite tetrahydrocannabinol carboxylic acid (THC-COOH). Most laboratories analyze Delta(9)THC in such samples using GC-MS methods, but these procedures are time-consuming and involve unavoidable previous extraction and derivatization. No data is yet available on the application of liquid chromatography-mass-spectrometry to detect Delta(9)THC in oral fluid. We report a validation method in which the Delta(9)THC is isolated from oral fluid by a simple liquid-liquid extraction with hexane and subsequently analyzed by liquid chromatography-mass-spectrometry. The method here reported for the determination of Delta(9)THC in oral fluid only requires 200 microl of sample and achieves limits of detection of 2 ng/ml, and has been used to analyze oral fluid samples collected from current drug users.     

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.     

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

The solution and solid state stability and excipient compatibility of parthenolide in feverfew

The objectives of this research were to evaluate the stability of parthenolide in feverfew solution state and powdered feverfew (solid state), and explore the compatibility between commonly used excipients and parthenolide in feverfew. Feverfew extract solution was diluted with different pH buffers to study the solution stability of parthenolide in feverfew. Powdered feverfew extract was stored under 40 degrees C/0% approximately 75% relative humidities (RH) or 31% RH/5~50 degrees C to study the influence of temperature and relative humidity on the stability of parthenolide in feverfew solid state. Binary mixtures of feverfew powered extract and different excipients were stored at 50 degrees C/ 75% RH for excipient compatibility evaluation. The degradation of parthenolide in feverfew solution appears to fit a typical first-order reaction. Parthenolide is comparatively stable when the environmental pH is in the range of 5 to 7, becoming unstable when pH is less than 3 or more than 7. Parthenolide degradation in feverfew in the solid state does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. After 6 months of storage, parthenolide in feverfew remains constant at 5 degrees C/31% RH. However, approximately 40% parthenolide in feverfew can be degraded if stored at 50 degrees C/31% RH. When the moisture changed from 0% to 75% RH, the degradation of parthenolide in feverfew increased from 18% to 32% after 6-month storage under 40 degrees C. Parthenolide in feverfew exhibits good compatibility with commonly used excipients under stressed conditions in a 3-week screening study.     

Reliability of urinary 6-sulfatoxymelatonin as a biomarker in breast cancer

The objective of this study is to evaluate the effect of cryopreservation at different storage temperatures on urinary 6-sulfatoxymelatonin (aMT6s) concentration. Overnight urine from 28 postmenopausal women participating in the ORDET cohort study was filtered and separated into 6 mL aliquots. Urine samples were stored at -80 degrees C and at -30 degrees C for an average of 14 years. Urinary aMT6s concentration was assessed using a competitive immunoassay. Mean aMT6s values of samples stored at -30 degrees C were systematically lower than those of samples stored at -80 degrees C (10.7 ng/mL versus 15.8 ng/mL, p<0.001). Bland Altman plots showed disagreement between determinations at different storage temperatures at the highest levels of the metabolite concentration. The degree of agreement evaluated in terms of intraclass correlation coefficient was 0.68 (95% CI 0.41-0.84, p<0.0001). Pearson's correlation coefficient between aMT6s values of the two differently stored samples was 0.93 (p<0.001), while the Kendal tau coefficient for rank distribution was 0.73 (p<0.001). Our data suggest that storage temperatures might affect degradation of aMT6s during storage. However, individual characterization by melatonin levels does not seem to be affected by cryopreservation conditions.     

Development of an Ointment Formulation Using Hot-Melt Extrusion Technology

Ointments are generally prepared either by fusion or by levigation methods. The current study proposes the use of hot-melt extrusion (HME) processing for the preparation of a polyethylene glycol base ointment. Lidocaine was used as a model drug. A modified screw design was used in this process, and parameters such as feeding rate, barrel temperature, and screw speed were optimized to obtain a uniform product. The product characteristics were compared with an ointment of similar composition prepared by conventional fusion method. The rheological properties, drug release profile, and texture characteristics of the hot-melt extruded product were similar to the conventionally prepared product. This study demonstrates a novel application of the hot-melt extrusion process in the manufacturing of topical semi-solids.     

Hydrothermal and pulp processing of Eucalyptus

Hydrothermal processing of Eucalyptus wood was performed at operation temperature of 181 degrees C, processing time or 37.5 min and solid water ratio of 1/6 to ensure a maximum loss of xylan recuperation with minimum cellulose fibre degradation. Under those conditions, the loss of xylan was 22% less than that achieved with the conditions 196 degrees C, 50.6 min and 1/8 (solid/water). IN In addition, an experimental design was used to study the influence of process variables: temperature (145-175 degrees C), pulping time (40-120 min) and ethanol concentration (40-70% weight concentration), on the properties of pulps (yield, kappa number, viscosity, cellulose, xylan, lignin acetyl groups contents and brightness) and paper sheets (stretch index, burst index and tear index) obtained from the solid fraction after hydrothermal treatment of Eucalyptus globulus. Pulps with acceptably high physical and chemical properties can be obtained operating at 175 degrees C for 90 min with 55% ethanol concentration.     

Measurement and modeling of multiple substrate oxidation by methanotrophs at 20 degrees C

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 degrees C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 degrees C to examine the effect of mixtures of trichloroethylene, trans-dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Delta model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 degrees C. Finally, it was also discovered that at 20 degrees C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 degrees C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.