Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Plasticity of death rates in stationary phase in Saccharomyces cerevisiae

For the species that have been most carefully studied, mortality rises with age and then plateaus or declines at advanced ages, except for yeast. Remarkably, mortality for yeast can rise, fall and rise again. In the present study we investigated (i) if this complicated shape could be modulated by environmental conditions by measuring mortality with different food media and temperature; (ii) if it is triggered by biological heterogeneity by measuring mortality in stationary phase in populations fractionated into subpopulations of young, virgin cells, and replicatively older, non-virgin cells. We also discussed the results of a staining method to measure viability instead of measuring the number of cells able to exit stationary phase and form a colony. We showed that different shapes of age-specific death rates were observed and that their appearance depended on the environmental conditions. Furthermore, biological heterogeneity explained the shapes of mortality with homogeneous populations of young, virgin cells exhibiting a simple shape of mortality in conditions under which more heterogeneous populations of older cells or unfractionated populations displayed complicated death rates. Finally, the staining method suggested that cells lost the capacity to exit stationary phase and to divide long before they died in stationary phase. These results explain a phenomenon that was puzzling because it appeared to reflect a radical departure from mortality patterns observed for other species.     

Stereoselective HPLC analysis of tertatolol in rat plasma using macrocyclic antibiotic chiral stationary phase

Enantiomeric resolution of teratolol was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase known as Chirobiotic V with UV detection set at 220 nm. The polar ionic mobile phase (PIM) consisted of methanol-glacial acetic acid-triethylamine (100:0.01:0.015, v/v/v) has been used at a flow rate of 0.8 ml min(-1) . The calibration curves in plasma were linear over the range of 5-500 ng ml(-1) for each enantiomer with detection limit of 2 ng ml(-1) . The proposed method was validated in compliance with the international conference on harmonization (ICH) guidelines. The developed method applied for the trace analyses of tertatolol enantiomers in plasma and for the pharmacokinetic study of tertatolol enantiomers in rat plasma. The assay proved to be suitable for therapeutic drug monitoring and chiral quality control for tertatolol formulations by HPLC.     

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH₄) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH₄ by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH₄ in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared with the parent strain, solely from CH₄. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH₄.     

Cellulose type chiral stationary phase based on reduced graphene oxide@silica gel for the enantiomer separation of chiral compounds

The graphene oxide (GO) was covalently coupled to the surfaces of silica gel (SiO₂) microspheres by amide bond to get the graphene oxide@silica gel (GO@SiO₂). Then, the GO@SiO₂ was reduced with hydrazine to the reduced graphene oxide@silica gel (rGO@SiO₂), and the cellulose derivatives were physically coated on the surfaces of rGO@SiO₂ to prepare a chiral stationary phase (CSP) for high performance liquid chromatography. Under the optimum experimental conditions, eight benzene‐enriched enantiomers were separated completely, and the resolution of trans‐stilbene oxide perfectly reached 4.83. Compared with the blank column of non‐bonded rGO, the separation performance is better on the new CSP, which is due to the existence of rGO to produce special retention interaction with analytes, such as π‐π stacking, hydrophobic effect, π‐π electron‐donor–acceptor interaction, and hydrogen bonding. Therefore, the obtained CSP shows special selectivity for benzene‐enriched enantiomers, improves separation selectivity and efficiency, and rGO plays a synergistic effect with cellulose derivatives on enantioseparation.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Analysis of benidipine enantiomers in human plasma by liquid chromatography-mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column

We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1 ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction-monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C(max) and AUC(inf) values of (+)-alpha benidipine were higher than those of (-)-alpha benidipine by 1.96- and 1.85-fold, respectively (p<0.001), whereas, the T(max) and t(1/2) for each of the benidipine stereoisomers were not significantly different.     

Diphenylethanediamine (DPEDA) as chiral selector: VII. Efficient HPLC resolution of a series of underivatized diarylcarbinols on a chiral stationary phase based on C5-amide-bonded N-3,5-dinitrobenzoyl-DPEDA

Fast and efficient baseline separation of asymmetrically substituted diarylmethanols and 1,1-diarylethanols was achieved on an endcapped, amide-linked N-3,5-dinitrobenzoylated, (R, R)-1,2-diphenyl-1,2-ethanediamine-derived chiral stationary phase (CSP). Optimal enantioselectivities on this CSP were obtained using 1% 2-propanol in n-heptane as the mobile phase. Enantiorecognition was found to be governed by π-basicity and the substitution pattern of the aromatic substituents. © 1996 Wiley-Liss, Inc.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Estrogenic and progestogenic effects of hormonal contraceptives in relation to sexual behavior: insights into extended sexuality

Women's mating adaptations may vary between fertile and luteal phases, given different costs and benefits of sexual activity during each phase. Women's non-conceptive (“extended”) sexuality might function in the context of pair-bonding. The current studies examined associations between women's loyalty and faithfulness to their relationships and frequency of sexual intercourse in women using hormonal contraception. As predicted, in study 1 estimated levels (adjusted for potency) of both synthetic estrogen and progestin delivered to women moderated the association between women's loyalty/faithfulness to their partner and frequency of intercourse: as estradiol levels diminished, and progestin levels increased, women's loyalty/faithfulness became more positively associated with frequency of intercourse. Study 2 replicated these findings in a sample of women studied over a 12 week period. Results further support claims for a possible function of extended sexuality, and speak to hormonal mechanisms affecting it. They also have important methodological and applied implications.     

Facile production of mono-substituted urea side chains in solid phase peptide synthesis

A method is reported for the straightforward generation of urea-containing peptides during Boc solid phase peptide synthesis. Primary amine side chains are converted to mono-alkyl ureas in two steps via an intermediate p-nitrophenyl carbamate. Use of p-methoxybenzyl amine as an ammonia equivalent affords mono-alkyl final products from standard resin cleavage methods, without the need for additional steps. The reaction is highly efficient and applicable to variable length side chains and peptides.     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

Chiral stationary phases in HPLC for the stereoselective determination of methadone

An extensive study of the behavior of three chiral stationary phases (CSP) used in liquid chromatography (LC) is presented for the stereoselective determination of methadone. The following chromatographic columns were selected: a cellulose, Chiralcel OJ; a modified cyclodextrin, Cyclobond I 2000 RSP, and a protein, Chiral‐AGP. Retention factors, enantioselectivity, efficiency, and resolution were tested by modifying the composition of the mobile phase as well as the temperature. The mechanism for the chiral recognition of methadone on each support was discussed. Optimal chromatographic parameters were obtained for the three supports tested, and methadone enantiomers were separated in less than 20 minutes. The cellulose‐based column gave the best resolution, but this CSP was not adapted to clinical analyses of methadone. Under optimized conditions, the cyclodextrin‐ and protein‐based columns allowed an excellent separation of methadone enantiomers, but no interference with the primary metabolite was found only with Chiral‐AGP. Chirality 11:319–325, 1999. © 1999 Wiley‐Liss, Inc.     

Solid-phase synthesis of chiral stationary phases based on 2,4,5,6-tetrachloro-1,3-dicyanobenzene derivatives spaced from N-3,5-dinitrobenzoyl alpha-amino acids: comparative study of their resolution efficacy

Two new chiral stationary phases, 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-D-phenylglycinyl) aminoethyl]aminophen-1-yl] aminopropyl silica (CSP-1) and 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-L-leucinyl) aminoethyl] aminophen-1-yl] aminopropyl silica (CSP-2), were prepared by solid-phase synthesis. They comprise chiral unit, 3,5-dinitrobenzoyl derivative of the amino acid, D-PhGly or L-Leu, bound via spacer 1,2-diaminoethane to 2,4-positions of the persubstituted benzene ring, derived from compound 1, and possess pseudo-C2 symmetry. Preparation of model compounds 6 and 7 confirmed the structure of chiral selectors, which comprise pi-donor persubsituted aromatic ring and two strong pi-acceptor 3,5-dinitrobenzoyl amido units. CD spectra of model selectors 6 and 7, run in DMSO above 250 nm, exhibit negative exciton coupling (EC) between pi-acceptor and pi-donor chromophores, C(1) symmetric model compound 8 exhibited much weaker EC and 9, devoid of pi-donor unit, does not exhibit any significant CD. Combined pi-donor and pi-acceptor properties enable the new CSPs to separate a broad range of racemates. The columns with CSP-1 and CSP-2 were tested for the separation of 22 racemates by HPLC with two different mobile phase systems and the results are compared with those obtained by using a structurally related commercial column.     

Determination of the enantiomeric purity of (−−) 2-(N-propyl-n-2-thienylethylamino)-5-hydroxytetralin (n-0923) by chiral stationary phase HPLC

The determination of the enantiomeric impurity, i.e., the percentage of (+) N?0437 (= N?0924) in several batches of (??) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.     

Optical resolution of glycerin sulfides and glycerin selenides on the chiral stationary phase of cellulose‐tris(3,5‐dimethylphenylcarbamate)

Cellulose‐tris(3,5‐dimethylphenylcarbamate) was prepared after a reported method and was coated onto an aminopropylated mesopore spherical silica gel. The final product was used as a chiral stationary phase of high performance liquid chromatography for the enantioseparation of a series of glycerin sulfides and glycerin selenides. Mixtures of hexane and 2‐propanol were used as mobile phases. The effects of 2‐propanol concentration in the mobile phase on the retention and resolution were investigated. Some enantiomers of the glycerin monosulfides and monoselenides could be separated satisfactorily, but none of the disulfides could be separated. The structural features of the solutes that influence chiral separation were discussed. Chirality 11:598–601, 1999. © 1999 Wiley‐Liss, Inc.     

Chromatographic resolution of the chiral isomers of several beta-blockers over cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

The optical resolution of seven beta-blockers which have in common the N-isopropyl-3-aryloxy-2-hydroxypropylamine moiety was carried out by HPLC using the cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase to quantitatively characterize the enantioselectivity of these compounds. The capacity factors and separation factors at different column temperature were determined with some qualitative trends derived. A compensation effect was observed for these compounds where there exists an approximately linear relationship between the enantiomeric differences in enthalpic and entropic energies.