ADI1, a methionine salvage pathway enzyme,is required for Drosophila fecundity

Background

Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity.

Results

Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity.

Conclusions

In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.     

Convenient method of threonine, methionine and their related amino compounds by high-performance liquid chromatography and its application to rumen fluid

A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO₂), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150×4.6 mm I.D., 5 μm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (μM) (S/N ratio, 3:1) and quantification (μM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO₂, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93–102.3% in the within-day study and 94.52–98.69% on different day (6 days) studies. The average contents (μM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency.     

甲硫氨酸限制对骨肉瘤细胞增殖、凋亡及铁死亡的影响

摘要 目的：探讨甲硫氨酸限制对骨肉瘤细胞增殖,凋亡和铁死亡的影响。方法：使用细胞计数检测剥夺甲硫氨酸对三株不同的骨肉瘤细胞系增殖的影响;高通量测序（RNA-seq）分析甲硫氨酸限制后骨肉瘤细胞的转录组学变化;流式细胞术检测细胞周期,凋亡以及ROS,脂质ROS水平;Western blot检测铁死亡关键蛋白谷胱甘肽过氧化物GPX4以及铁死亡标志蛋白前列腺素内过氧化物合成Cox-2的表达。结果：甲硫氨酸限制显著抑制骨肉瘤细胞的增殖（P<0.001）;RNA-seq分析筛选出1719个差异表达基因,基因富集分析发现甲硫氨酸限制可显著激活铁死亡通路并显著抑制细胞周期通路;细胞实验证实甲硫氨酸限制将骨肉瘤细胞阻滞在G2M期,显著诱导凋亡细胞比例增加;同时,细胞内Cox-2的表达增加,GPX4的活性降低,活性氧和脂质活性氧积累,最终导致细胞死亡,且这种作用可以被铁死亡抑制剂Fer-1部分挽救。结论：本研究的结果为甲硫氨酸限制治疗骨肉瘤提供了一定的科学依据。     

Molecular cloning,expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC6803

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

天然氨基酸诱导野油菜黄单胞菌降解DSF-家族群体感应信号活性分析

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris,Xcc)是十字花科植物黑腐病的致病菌。Xcc中DSF (Diffusible signal factor)信号依赖的群体感应系统和RpfB介导的群体感应退出机制均与其致病性密切相关。【目的】分别检测18种氨基酸对DSF-家族群体感应信号分子合成的影响,为研发新型生物防治方法提供思路。【方法】添加不同浓度的氨基酸到ΔrpfC菌株XYS培养体系中,接种后不同时间点取样提取DSF信号分子,利用高效液相色谱法(High performance liquid chromatography,HPLC)分析DSF和BDSF浓度。【结果】18种氨基酸中,甲硫氨酸、色氨酸和胱氨酸能有效降低ΔrpfC菌株培养体系中DSF和BDSF水平,抑制效果与氨基酸浓度密切相关;3种氨基酸对DSF信号分子的抑制作用存在叠加效应;甲硫氨酸、色氨酸或胱氨酸不影响ΔrpfCΔrpfB双突变体菌株中DSF和BDSF水平。【结论】首次发现了甲硫氨酸、色氨酸和胱氨酸通过RpfB诱导Xcc退出群体感应状态。     

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of ³H-labelled [DAla²]-[Dleu⁵]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control. On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [³H]dihydromorphine and [³H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To wval;uate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 μM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligan-receptor recognition. Whether this can offer a satisfactory explanation for our obervations remains to be established.     

Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone

The present study attempts to compile information on the possible physiologic role of the endogenous peptide neurotensin (NT) as a hormone and/or neurotransmitter. The methodological approach is immunohistochemical localization of NT in the entero-endocrine system as well as in the central and peripheral nervous systems. The results found in the three systems are first related to the pharmalogical and physiological findings in the literature. Subsequently their significance is discussed for each organ separately before attempting a final overall interpretation. Briefly, the present study reveals the following essential findings: The occurrence and distribution of NT-IR entero-endocrine cells (N-cells) in different mammals including man, as well as in representative members of all classes of vertebrates and higher invertebrates, are analyzed and evaluated morphometrically. The NT-IR cells in all investigated species are demonstrated to be of the open type. The innervation of paravertebral and prevertebral ganglia by NT-IR fibers is described; at least a portion of these fibers is thought to originate in NT-IR perikarya of the substantia intermedia of the spinal cord. The involvement of these NT-IR fibers in the regulation of systemic blood flow (hypertension) is suggested. The existence of NT-IR innervation of the gastro-intestinal tract is considered to be a general phenomenon. This notion is reaffirmed by phylogenetic investigation of the NT-IR enteric nerves. The pharmacological effects of NT in different portions of the gastro-intestinal tract, reported in the literature are related to the immunohistochemical localization of NT. In light of the present results, some of the effects of NT which were previously considered to be of an endocrine or paracrine nature - such as contraction of the guinea-pig ileum - are interpreted as effects of NT of neuronal origin. The specific NT-IR innervation of target cells in the exocrine pancreas (vascular smooth muscle, acinar cells) is demonstrated, and participation of NT-IR nerve fibers in regulation of the secretion of pancreatic juice is postulated. The innervation of the heart (coronary vasculature, myocardium, conduction system) by NT-IR fibers is demonstrated in various mammals and for the first time also in man. The cardiac NT-IR nerve fibers are thought to be the cytological substrate for different NT effects on heart action (coronary vasoconstriction, positive inotropy and chronotropy) reported in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Betaine lipids

Diacylglyceryltrimethylhomoserine (DGTS) and diacylglyceryl hydroxymethyltrimethyl-β-alanine (DGTA) belong to a new type of glycerolipids called betaine lipids, which are composed of diacylglycerol andN-permethylated hydroxyamino acids, that are linked by an ether linkage. Betaine lipids are widely distributed in lower plants and algae as well as in some non-photosynthetic microorganisms. They are no longer regarded as “unusual” lipids but are important constituents of membranes of lower organisms. The present state of knowledge of the phylogenetic distribution, the fatty acid composition, the thermal properties, and the biosynthesis of the bataine lipids is briefly summarized in this review in a perspective of a future search for the biological roles and activities of betaine lipids. Recipient of the Botanical Society Award of Young Scientists, 1991.