Progress toward re‐engineering non‐ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Many important pharmaceutical agents, including vancomycin, bleomycin, cyclosporin, and several antibiotics, are produced by non‐ribosomal peptide synthetase (NRPS) enzymes in microorganisms. The NRPS pathway produces an extensive library of products using multienzyme complexes acting in an assembly‐line fashion. Engineering an NRPS system to produce an even greater variety of products, some of which may also have beneficial therapeutic value, would be an enormous advantage. Several approaches have been successful in generating novel NRPS products: mutational biosynthesis during which nonnatural substrates are fed to an organism; domain and module swapping between different species to generate hybrid enzymes; and rational site‐directed mutagenesis, based either on phylogeny or computational prediction, intended to switch substrate specificity and produce altered products. This review will highlight the progress in these areas and describe research in the future that will extend the capacity for re‐engineering NRPS systems. Drug Dev. Res. 66:9–18, 2006. © 2006 Wiley‐Liss, Inc.     

萘普生对大鼠着床期宫内PGF_(2α)含量及宫内节育器抗生育作用的影响

本文测定了对照组及两个萘普生(前列腺素合成酶抑制剂)处理组大鼠着床期宫内PGF2α的含量。结果表明,对照组、萘普生低剂量组(2mg/kg体重)和高剂量组(20mg/kg体重)的PGF2α含量分别为6.71±0.93、2.19±0.34和1.01±0.18ng/100g组织(三组间P<0.01)。同时,还观察到在本实验条件下,萘普生无明显干扰节育器抗生育作用,药物本身亦未呈现显著抗生育活性。     

Localization of glial cell antigens in the brains of young normal mice and the dysmyelinating mutant mice, jimpy and shiverer

Tissue sections from the brains of normal, jimpy, and shiverer mice were immunostained by the peroxidase antiperoxidase method for carbonic anhydrase (CA) and the putative astrocytic "markers" glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The cells in normal gray matter that immunostained with anti-CA and anti-GS were similar to one another in size and process elaboration. In the normal gray matter there were relatively few GFAP-positive astrocytes. When present, these cells resembled the CA- and GS-positive cells; however, the GFAP appeared to be concentrated in the astroglial processes, as distinguished from the cell bodies. Glial cell processes, immunostained for CA or GS, surrounded blood vessels and unstained neurons in the normal gray matter. The glial cells in shiverer gray matter were similar to those in the normal gray matter. When stained for GS or GFAP, the glial cells in the jimpy gray matter appeared to be somewhat hypertrophied, and when the glial cells in this mutant were stained for CA, the nuclei appeared to be swollen. It was concluded that some of the CA-positive cells in the gray matter of the normal and of each mutant mouse brain could be astrocytes. The patterns of immunostaining in the white matter emphasized the different complements of glial cells in the mutants. In the normal and shiverer mouse corpus callosum, CA, in particular, was detected only in the oligodendrocytes, their processes, and myelin. However, the data concerning the jimpy mouse suggested that the few CA-positive cells in the corpus callosum of that mutant could be astrocytes.     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Isolation of a DNA fragment which complements glutamine synthetase deficient strains ofS. pombe

Summary From a gene bank ofS. pombe DNA, a 5.6 kb clone was isolated which complemented mutants defective in glutamine synthetase (GS) activity. Sub-cloning fragments of this 5.6 kb clone showed that the complementing activity was localised in a 1.6 kb HindIII-Aval fragment and a partial DNA sequence revealed an open reading frame preceded by TATA sequences and a TGACTA sequence. Plasmid constructs carrying up to 3.4 kb of DNA used to transformgln ⁻ strains gave transformants which showed a wide range of GS activity, in some cases 100 times the wild-type level. These constructs identify DNA sequences lying downstream from the putative coding sequence which have effects on the total amount of enzyme activity, but do not affect the control imposed by the nitrogen source on which the cells are grown.     

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.     

转化生长因子β1对香烟诱导的大鼠肺泡巨噬细胞γ-谷氨酰半胱氨酸合酶表达的影响

目的：探讨转化生长因子（TGF）-β1对香烟诱导的大鼠肺泡巨噬细胞γ-谷氨酰半胱氨酸合酶（γ-GCS）和活化蛋白(AP)-1亚单位c-fos mRNA及其蛋白表达的影响。方法:通过支气管肺泡灌洗获取大鼠肺泡巨噬细胞，随机分为对照组、香烟组和TGF-β1组。TGF-β1组加入终浓度为3 μg/L的TGF-β1,2 h后除对照组外，余2组均加入香烟烟雾提取物,对照组加入磷酸盐缓冲液。分别用逆转录聚合酶链式反应（RT-PCR）和免疫细胞化学方法检测肺泡巨噬细胞中γ-GCSh(重链亚单位)及c-fos mRNA和蛋白的表达。结果:香烟组γ-GCSh和c-fos mRNA及其蛋白表达水平显著高于对照组(分别P<0.05,P<0.01)。TGF-β1组γ-GCSh mRNA及其蛋白表达水平明显低于香烟组(均P<0.01)；而c-fos mRNA及其蛋白表达水平明显高于香烟组(均P<0.01)。结论:转化生长因子-β1参与香烟所致慢性阻塞性肺疾病肺氧化/抗氧化失衡的发病机制。     

Protective effect of 3-aminobenzamide,an inhibitor of poly (ADP-ribose) synthetase,against streptozotocin-induced diabetes

Summary The addition of 3-aminobenzamide (a potent inhibitor of poly(ADP-ribose)synthetase) into the incubation medium, prevents streptozotocin-induced inhibition of glucose-stimulated insulin release from isolated islets [control 142±14U·islet^–1·h^–1; streptozotocin (0.5mg/ml) 31±8; 3-aminobenzamide (l.0 mg/ml) 96±11; streptozotocin plus 3-aminobenzamide 122±19]. In vivo, intraperitoneal 3-aminobenzamide 300 mg/kg body weight prevents the appearance of overt diabetes in streptozotocin-treated rats. These protective effects of 3-aminobenzamide are dose-dependent and are similar to those exerted by nicotinamide. Taking into account that poly ADP-ribosylation is involved in the repair of damaged DNA, the protection exerted by 3-aminobenzamide against the diabetogenic effect of streptozotocin strongly supports the view that this acute effect may be a major consequence of the activation of DNA repair mechanisms in islet cells.