Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5′ or 3′ cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients’ fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of ¹⁴C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.     

Methylmalonyl acidemia: from mitochondrial metabolism to defective mitophagy and disease

ABSTRACT

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism due to the deficiency of mitochondrial MMUT (methylmalonyl-CoA mutase) – an enzyme that mediates the cellular breakdown of certain amino acids and lipids. The loss of MMUT leads to the accumulation of toxic organic acids causing severe organ dysfunctions and life-threatening complications. The mechanisms linking MMUT deficiency, mitochondrial alterations and cell toxicity remain uncharacterized. Using cell and animal-based models, we recently unveiled that MMUT deficiency impedes the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, thereby halting their delivery and subsequent degradation by macroautophagy/autophagy-lysosome systems. In turn, this defective mitophagy process instigates the accumulation of dysfunctional mitochondria that spark epithelial distress and tissue damage. Correction of PINK1-directed mitophagy defects or mitochondrial dysfunctions rescues epithelial distress in MMA cells and alleviates disease-relevant phenotypes in mmut?deficient zebrafish. Our findings suggest a link between primary MMUT deficiency and diseased mitochondria, mitophagy dysfunction and cell distress, offering potential therapeutic perspectives for MMA and other metabolic diseases.     

Two Frequent Mutations Associated with the Classic Form of Propionic Acidemia in Taiwan

Propionyl-CoA carboxylase (PCC) is involved in the catabolism of branched chain amino acids, odd-numbered fatty acids, cholesterol, and other metabolites. PCC consists of two subunits, α and β, encoded by the PCCA and PCCB genes, respectively. Mutations in the PCCA or PCCB subunit gene may lead to propionic acidemia. In this study, we performed mutation analysis on ten propionic acidemia patients from eight unrelated and nonconsanguineous families in Taiwan. Two PCCA mutations, c.229C→T (p.R77W) and c.1262A→C (p.Q421P), were identified in a PCCA-deficient patient. Six mutations in the PCCB gene, including c.-4156_183+3713del, c.580T→C (p.S194P), c.838dup (p.L280Pfs*11), c.1301C→T (p.A434V), c.1316A→G (P.Y439C), and c.1534C→T (p.R512C), were identified in seven PCCB-deficient families. The c.-4156_183+3713del mutation is the first known large deletion that affects the PCCB gene functions. Furthermore, the c.1301C→T and c.-4156_183+3713del mutations in the PCCB gene have not been reported previously. Clinical features demonstrated that these two frequent mutations are associated with low enzyme activity and a classic propionic acidemia phenotype.     

Quantitative analysis of mitochondrial protein expression in methylmalonic acidemia by two-dimensional difference gel electrophoresis

Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA.     

Novel Mutations Found in Two Genes of Thai Patients with Isolated Methylmalonic Acidemia

Molecular genetic analysis of three patients diagnosed with isolated methylmalonic acidemia (MMA) revealed that one was mut ⁰ MMA, with a mutation in the MUT gene encoding the l-methylmalonyl-CoA mutase (MCM), and two were cblB MMA, with mutations in the MMAB gene required for synthesizing the deoxyadenosylcobalamin cofactor of MCM. The mut ⁰ patient was homozygous for a novel nonsense mutation in MUT, p.R31X (c.167C → T), and heterozygous for three previously described polymorphisms, p.K212K (c.712A → G), p.H532R (c.1671A → G), and p.V671I (c.2087G → A). The new MMAB mutation, p.E152X (c.454G → T), was found to be homozygous in one cblB patient and heterozygous in the other patient, who also had four intron polymorphisms in this gene.     

Development of simple and effective PCR based assay to detect PCCA mutation (c.425G > A) among Saudi carriers and functional study of the homozygous PCCA mutations

The aim of this study is to develop a rapid and effective method to screen for Saudi carriers of one of the most common propionic acidemia mutations (c.425G > A) and to study the functional impact of this mutation. Using allele-specific primers, we have developed a qPCR assay that clearly distinguishes heterozygotes from mutated and wild type homozygotes that overcome the dependence on labor-intensive gene sequencing. We show here that (i) qPCR rapid test has strong accuracy in detecting (c.425G > A) mutation in heterozygotes and homozygotes individuals and that the Ct-value cut-offs were estimated to be and 23.37 ± 0.04 (CV-6 %, 95 %CI-7.25) for homozygote, 25.06 ± 0.02 (CV-3.5 %, 95 %CI-7.85) for heterozygote PCCA c.425G > A mutation and 29.55 ± 0.002 (CV-11 %, 95 %CI-1.41) for PCCA wild type; (ii) the incidence of PA heterozygotes/carriers in Saudi population is about 550/100,000; (iii) skin fibroblast assays show that homozygote c.425G > A mutation induced propionyl-CoA carboxylase activity abrogation, (iv) PA patients showed an increased level of propionyl carnitine C3 in blood and 3-hydroxy propionic acid and methyl citrate in urine. Conclusion: qPCR represent an effective strategy to assess for PCCA mutation carriers in the Saudi population and we believe that will help in preventing homozygosity in the population after been implemented in pre-marriage screening program.     

Structural investigation and inhibitory response of halide on phosphoserine aminotransferase from Trichomonas vaginalis

BackgroundPhosphoserine aminotransferase (PSAT) catalyses the second reversible step of the phosphoserine biosynthetic pathway in Trichomonas vaginalis, which is crucial for the synthesis of serine and cysteine.MethodsPSAT from T. vaginalis (TvPSAT) was analysed using X-ray crystallography, enzyme kinetics, and molecular dynamics simulations.ResultsThe crystal structure of TvPSAT was determined to 2.15 Å resolution, and is the first protozoan PSAT structure to be reported. The active site of TvPSAT structure was found to be in a closed conformation, and at the active site PLP formed an internal aldimine linkage to Lys 202. In TvPSAT, Val 340 near the active site while it is Arg in most other members of the PSAT family, might be responsible in closing the active site. Kinetic studies yielded Km values of 54 μM and 202 μM for TvPSAT with OPLS and AKG, respectively. Only iodine inhibited the TvPSAT activity while smaller halides could not inhibit.ConclusionResults from the structure, comparative molecular dynamics simulations, and the inhibition studies suggest that iodine is the only halide that can bind TvPSAT strongly and may thus inhibit the activity of TvPSAT.The long loop between β8 and α8 at the opening of the TvPSAT active site cleft compared to other PSATs, suggests that this loop may help control the access of substrates to the TvPSAT active site and thus influences the enzyme kinetics.General significanceOur structural and functional studies have improved our understanding of how PSAT helps this organism persists in the environment.     

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

磷酸丝氨酸氨基转移酶1介导肺腺癌细胞粘附及其机制探究

摘要 目的：探究丝氨酸生物合成途径（SSP）关键酶磷酸丝氨酸氨基转移酶1（PSAT1）与肺腺癌细胞粘附的关系，并初步探讨其作用机制。方法：使用siRNA抑制PSAT1蛋白表达，观察肺腺癌细胞形态以及粘附变化，同时过表达PSAT1，反向观察PSAT1对肺腺癌细胞粘附的影响。初步探究其作用机制，采用免疫共沉淀-蛋白质谱法寻找与PSAT1直接相互作用的蛋白，筛选差异蛋白，并在过表达细胞体系中验证。结合临床公共数据库分析互作蛋白与患者预后关系。结果：发现敲低PSAT1引起肺腺癌细胞形态改变；敲低PSAT1抑制肺腺癌PC9、HCC827细胞粘附；过表达PSAT1增强PC9及HCC827细胞粘附；免疫共沉淀-蛋白质谱检测到2560个可能与PSAT1结合的蛋白，进一步通过免疫共沉淀-免疫印迹法验证PSAT1过表达使细胞中与间皮素（MSLN）结合显著上升；通过临床样本数据观察PSAT1与MSLN共同高表达的肺腺癌患者，其预后更差。结论：本文首次报道PSAT1可能通过与MSLN等蛋白-蛋白相互作用影响肺腺癌细胞粘附的新机制，提示PSAT1有望成为潜在抗肿瘤靶点，靶向其相互作用蛋白能为小分子抑制剂设计及患者个体化治疗提供新思路。     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products

Background aimsChimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells.MethodsWe used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency.ResultsResults indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells.ConclusionsThese studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.     

Role of the microsomal mixed-function amine oxidase in the oxidation of N,N-disubstituted hydroxylamines

S-Adenosylhomocysteine inhibits betaine-homocysteine methyltransferase. The inhibition is nonlinear, competitive in relation to homocysteine, and noncompetitive in relation to betaine. S-Adenosylhomocysteine activates cystathionine synthase at all concentrations of the substrates, serine and homocysteine. By altering the distribution of homocysteine between these competing pathways, S-adenosylhomocysteine may be significant in the regulation of methionine metabolism in the intact animal.     

Organic acid profile of isovaleric acidemia: a comprehensive metabolomics approach

Isovaleric acidemia (IVA, MIM 248600) can be a severe and potentially life-threatening disease in affected neonates, but with a positive prognosis on treatment for some phenotypes. This study presents the first application of metabolomics to evaluate the metabolite profiles derived from urine samples of untreated and treated IVA patients as well as of obligate heterozygotes. All IVA patients carried the same homozygous c.367 G > A nucleotide change in exon 4 of the IVD gene but manifested phenotypic diversity. Concurrent class analysis (CONCA) was used to compare all the metabolites from the original complete data set obtained from the three case and two control groups used in this investigation. This application of CONCA has not been reported previously, and is used here to compare four different modes of scaling of all metabolites. The variables important in discrimination from the CONCA thus enabled the recognition of different metabolic patterns encapsulated within the data sets that would not have been revealed by using only one mode of scaling. Application of multivariate and univariate analyses disclosed 11 important metabolites that distinguished untreated IVA from controls. These included well-established diagnostic biomarkers of IVA, endogenous detoxification markers, and 3-hydroxycaproic acid, an indicator of ketosis, but not reported previously for this disease. Nine metabolites were identified that reflected the effect of treatment of IVA. They included detoxification products and indicators related to the high carbohydrate and low protein diet which formed the hallmark of the treatment. This investigation also provides the first comparative metabolite profile for heterozygotes of this inherited metabolic disorder. The detection of informative metabolites in even very low concentrations in all three experimental groups highlights the potential advantage of the holistic mode of analysis of inherited metabolic diseases in a metabolomics investigation.     

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.     

丝氨酸生物合成途径介导肺腺癌细胞EGFR-TKIs靶向治疗适应性耐药

目的:研究丝氨酸生物合成途径(SSP)在肺腺癌使用表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)治疗后引起的适应性耐药中发挥的作用,探究早期适应性耐药机制以寻找抗耐药靶标。方法:使用EGFR-TKIs药物短时刺激肺腺癌细胞系后,利用Western blotting和qRT-PCR技术检测丝氨酸生物合成途径中关键酶的蛋白及m RNA水平变化,同时利用LC-MS检测细胞内丝氨酸生物合成途径产物及相关代谢产物变化情况。通过CCK8法检测敲低关键酶对细胞增殖的影响。体内实验进行肺腺癌细胞裸鼠皮下移植瘤注射,采用剂量爬坡法构建体内适应性耐药模型,检测肿瘤组织中关键酶表达情况。结果:1.细胞内丝氨酸生物合成途径关键酶PHGDH、PSAT1、PSPH的蛋白表达水平在不同药物作用时间和浓度下有不同程度上调,且m RNA水平也上调了20-50%左右(P0.05);2.HCC827细胞中SSP及下游代谢通路产物如P-Serine、Serine、Glycine、AMP等均有显著性上调(P0.01);3.敲低关键酶PSAT1及PSPH后可抑制细肺腺癌细胞HCCC827及PC9的增殖,与对照组相比最高抑制率可达60%左右(P0.01);4.体内诱导PC9细胞适应性耐erlotinib后,肿瘤组织中的PHGDH及PSAT1表达均有明显上调。结论:丝氨酸生物合成途径介导了肺腺癌EGFR-TKIs靶向治疗的适应性耐药,其关键酶有望作为抗耐药靶标进行联合治疗,从而提高EGFR-TKIs靶向药物的早期疗效并最终克服耐药性的产生。     

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.     

Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients’ cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls’ fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

The role of serine in the lipid metabolism of the rat tapeworm Hymenolepisdiminuta

Webb R. A. and Mettrick D. F. 1973. The role of serine in the lipid metabolism of the rat tapeworm Hymenolepis diminuta. International Journal for Parasitology3: 47–58. The inter-relationship between the amino acid serine and lipid metabolism in the rat tapeworm Hymenolepis diminuta has been studied under in vitro conditions. The label from U-¹⁴C-serine, U-¹⁴C-glucose and 1-¹⁴C-oleic acid was rapidly incorporated into worm tissue phospho- and glycolipids, the latter illustrating the synthesis of cerebrosides by H. diminuta. Activity from U-¹⁴C-serine was recovered in phosphatidylserine, phosphatidylethanolamine, cerebrosides and several unidentified lipid-like compounds. The majority of the label recovered in phosphatidylethanolamine was associated with the ethanolamine moiety; in the cerebrosides with the sphingosine moiety. The sugar moiety of the cerebrosides was galactose.Pulse label studies showed a serine flux phenomenon, and a rapid rate of turnover of some of the unidentified compounds.Exogenous ethanolamine had no detectable effect upon absorption and conversion of serine to tissue phosphatidylethanolamine. Incubation of H. diminuta homogenates with phosphatidyl U-¹⁴C-serine resulted in the recovery of considerable activity in phosphatidylethanolamine. The results show that the major pathway of phosphatidylethanolamine synthesis is by decarboxylation of phosphatidylserine.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.