PBPK-PD model for predicting morphine pharmacokinetics, CNS effects and naloxone antagonism in humans.

Morphine and morphine-6-glucuronide (M6G) produce central nervous system (CNS) effects by activating mu-opioid receptors, while naloxone is used mainly for the reversal of opioid overdose, specifically for the fatal complication of respiratory depression, but also for alleviating opioid-induced side effects. In this study we developed a physiologically-based pharmacokinetic-pharmacodynamic (PBPK-PD) model to simultaneously predict pharmacokinetics and CNS effects (miosis, respiratory depression and analgesia) of morphine as well as antagonistic effects of naloxone against morphine. The pharmacokinetic and pharmacodynamic parameters were obtained from in vitro data, in silico, or animals. Pharmacokinetic and pharmacodynamic simulations were conducted using 39 and 36 clinical reports, respectively. The pharmacokinetics of morphine and M6G following oral or intravenous administration were simulated, and the PBPK-PD model was validated using clinical observations. The Emax model correlated CNS effects with free concentrations of morphine and M6G in brain parenchyma. The predicted CNS effects were compared with observations. Most clinical observations fell within the 5th-95th percentiles of simulations based on 1000 virtual individuals. Most of the simulated area under the concentration-time curve or peak concentrations also fell within 0.5-2-fold of observations. The contribution of morphine to CNS effects following intravenous or oral administration was larger than that of M6G. Pharmacokinetics and antagonistic effects of naloxone on CNS effects were also successfully predicted using the developed PBPK-PD model. In conclusion, the pharmacokinetics and pharmacodynamics of morphine and M6G, antagonistic effects of naloxone against morphine-induced CNS effects may be successfully predicted using the developed PBPK-PD model based on the parameters derived from in vitro, in silico, or animal studies.

TA, GT and AC are significantly under-represented in open reading frames of prokaryotic and eukaryotic protein-coding genes.

Genomes can be considered a combination of 16 dinucleotides. Analysing the relative abundance of different dinucleotides may reveal important features of genome evolution. In present study, we conducted extensive surveys on the relative abundances of dinucleotides in various genomic components of 28 bacterial, 20 archaean, 19 fungal, 24 plant and 29 animal species. We found that TA, GT and AC are significantly under-represented in open reading frames of all organisms and in intergenic regions and introns of most organisms. Specific dinucleotides are of greatly varied usage at different codon positions. The significantly low representations of TA, GT and AC are considered the evolutionary consequences of preventing formation of pre-mature stop codons and of reducing intron-splicing options in candidate primary mRNA sequences. These data suggest that a reduction of TA and GT occurred on both strands of the DNA sequence at an early stage of de novo gene birth. Interestingly, GT and AC are also significantly under-represented in current prokaryotic genomes, suggesting that ancient prokaryotic protein-coding genes might have contained introns. The greatly varied usages of specific dinucleotides at different codon positions are considered evolutionary accommodations to compensate the unavailability of specific codons and to avoid formation of pre-mature stop codons. This is the first report presenting data of dinucleotide relative abundance to indicate the possible existence of spliceosomal introns in ancient prokaryotic genes and to hypothesize early steps of de novo gene birth.

Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization.

Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5'-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5'-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories.

[Purification of Taq DNA polymerase expressed in Escherichia coli].

Taq DNA polymerase is one of the most commonly thermostable DNA polymerases in molecular biological researches, which shares its basic characters with others of the family, thereby its purifying strategy could be used not only in itself production but also in the extraction of the others as a reference. At present, the protocols reported for large scale preparation of Taq DNA are high cost, so a cheaper method was described here. In this protocol, by heat denaturation, ammonium sulfate precipitation and cation exchange chromatography of 724 resin, about 18 g powder of Na form resin could recover about 27.07 mg of Taq enzyme. The total activity and specific activity were approximately 2.2 × 105 U and 8131.98 U/mg. The total yield was about 48.92% with 59.35 of purification folds. Analysis of quality of purified enzyme indicated that only one protein 94 kDa was identified against SDS-PAGE and the remnant of DNA nuclease was not detected. For PCR reaction, The amplification ability of purified Taq polymerase was not different from that of the commercially avail-able ones. This method reported in the present study is effective and low cost, making it suitable for general purification in laboratories or business production.