Development of efficient suicide mechanisms for biological containment of bacteria

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.     

Aminergic neuron systems of lobsters: morphology and electrophysiology of octopamine-containing neurosecretory cells

In the American lobster (Homarus americanus) the biogenic amines serotonin and octopamine appear to play important and opposite roles in the regulation of aggressive behavior, in the establishment and/or maintenance of dominant and subordinate behavioral states and in the modulation of the associated postural stances and escape responses. The octopamine-containing neurosecretory neurons in the thoracic regions of the lobster ventral nerve cord fall into two morphological subgroups, the root octopamine cells, a classical neurohemal group with release regions along second thoracic roots, and the claw octopamine cells, a group that selectively innervates the claws. Cells of both subgroups have additional sets of endings within neuropil regions of ganglia of the ventral nerve cord. Octopamine neurosecretory neurons generally are silent, but when spontaneously active or when activated, they show large overshooting action potentials with prominent after-hyperpolarizations. Autoinhibition after high-frequency firing, which is also seen in other crustacean neurosecretory cells, is readily apparent in these cells. The cells show no spontaneous synaptic activity, but appear to be excited by a unitary source. Stimulation of lateral or medial giant axons, which excite serotonergic cells yielded no response in octopaminergic neurosecretory cells and no evidence for direct interactions between pairs of octopamine neurons, or between the octopaminergic and the serotonergic sets of neurosecretory neurons was found.     

Functional divergence of the circadian clock proteins in prokaryotes

Cyanobacteria are only prokaryotes known so far to have a circadian system. It may be based either on two (kaiB and kaiC) or three (kaiA, kaiB and kaiC) circadian genes. The homologs of two circadian proteins, KaiB and KaiC, form four major subfamilies (K1–K4) and also occur in some other prokaryotes. Using the likelihood-ratio tests, we studied a rate shift at the functional divergence of the proteins from the different subfamilies. It appears that only two of the subfamilies (K1 and K2) perform circadian functions. We identified in total 92 sites that have significantly different rates of evolution between the clades K1/K2 and K3/K4; 67 sites (15 in KaiB and 52 in KaiC) been evolving significantly slower in K1/K2 than the overall average for the entire sequence. Many critical sites are located in the identified functionally important motifs and regions, e.g. one of the Walker’s motif As, DXXG motif, and two KaiA-binding domains of KaiC. There are also 36 sites (~5%) with rate shift between K1 and K2. The rate shift at these sites may be related to the interaction with KaiA. Rate shift analyses have identified residues whose manipulation in the Kai proteins may lead to better understanding of their functions in the two different types of the cyanobacterial circadian system.     

A fast and versatile method for extraction and quantitation of long-chain acyl-CoA esters from tissue: content of individual long-chain acyl-CoA esters in various tissues from fed rat.

A method for the extraction of acyl-CoA esters from tissue, and their subsequent analysis by HPLC is described. The lipids are removed by a two-phase extraction in a chloroform/methanol/water system. The long-chain acyl-CoA esters are extracted using methanol and a high salt concentration (2 M ammonium acetate). Reextraction of the dry residue after evaporation of extraction solvent results in low overall recoveries (20%). By adding 1 mg/ml acyl-CoA-binding protein to the extraction solvent the overall recovery was increased to 55%. The method is easy and fast to perform and is thereby suitable for analysis of a large number of samples. The advantages of the method over previously published methods are discussed.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.