基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Nucleotide sequence of the R721 shufflon.

The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.     

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Analysis of nuclear 3,3',5-triiodothyronine receptor in the brown adipose tissue (BAT) of the postnatal lamb.

Postnatal thermogenesis in sheep is associated with increased sympathoadrenal activities, a T3 surge and an enhanced brown adipose tissue (BAT) type II 5'-monodeiodinating (5'-MDI) activity. The latter peaks 3-4 days after birth and is known to be important in generating intracellular T3 for nuclear receptor binding. In order to further investigate the mechanism(s) responsible for neonatal thermogenesis, thyroid hormone nuclear receptor (T3NR) binding characteristics were quantified in lamb BAT from newborn (NB) to 30d of postnatal age. Maximal binding capacities (MBC, mean +/- SEM fmoles T3/mg DNA) in BAT showed a decrease as studied by ANOVA during the first 11 days (NB to 1d, 148 +/- 24 [N = 5, p < 0.01, cf. 3-5d group]; 3-5 d, 61 +/- 5.5 [N = 5]; 10-11d, 72 +/- 9.1 [N = 4]). Afterwards, MBC increased at 30d (196 +/- 32, N = 4, p less than 0.01, cf. 3-5d group). BAT T3NR binding affinities (10(9) M-1) were comparable in all age groups studied (NB-1d, 2.8 +/- 0.3; 3-5d, 3.4 +/- 0.3; 10-11d, 4.0 +/- 1.1; 30d, 2.4 +/- 0.4). The data suggest that the postnatal surge in T3 and type II 5'-MDI is accompanied with a concurrent decrease in MBC of BAT T3NR. The latter may represent a down-regulation of T3NR presumably in an attempt to regulate the overall effect of thyroid hormone in neonatal thermogenesis.