Regulation of membrane phospholipid biosynthesis in mammalian cells.

In mammalian cells, phospholipids and cholesterol are assembled into bilayer membranes forming the plasma membrane, nuclear envelope, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and endosomes. Phospholipids are divided into classes based on the molecular structures, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, cardiolipin, and sphingomyelin. In addition to their structural roles, phospholipids play important roles in many cellular processes, such as membrane protein regulation, membrane trafficking, cell growth, apoptosis, and intracellular signaling. Thus, abnormal phospholipid metabolism is associated with various diseases. In mammalian cells, phospholipid classes are generated through several enzymatic steps, predominantly in the endoplasmic reticulum, mitochondria, and Golgi apparatus. In recent years, various enzymes involved in the biosynthesis of phospholipid classes have been identified. However, little is known about the regulatory mechanisms underlying the biosynthesis of phospholipid classes. Using our recently developed enzymatic fluorometric assays for all major phospholipid classes, we have demonstrated changes in phospholipid composition in intracellular organelles during cell growth. In this review, we summarize the current understanding of the properties and functions of phospholipid biosynthesis enzymes, and discuss their regulatory mechanisms

Application of enzymatic fluorometric assays to quantify phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in human plasma lipoproteins.

Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5-2.8 %, 1.1-2.4 % and 0.9-2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7-4.7 %, 2.1-4.5 % and 1.6-3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins

Alterations in cellular and organellar phospholipid compositions of HepG2 cells during cell growth.

The human hepatoblastoma cell line, HepG2, has been used for investigating a wide variety of physiological and pathophysiological processes. However, less information is available about the phospholipid metabolism in HepG2 cells. In the present report, to clarify the relationship between cell growth and phospholipid metabolism in HepG2 cells, we examined the phospholipid class compositions of the cells and their intracellular organelles by using enzymatic fluorometric methods. In HepG2 cells, the ratios of all phospholipid classes, but not the ratio of cholesterol, markedly changed with cell growth. Of note, depending on cell growth, the phosphatidic acid (PA) ratio increased and phosphatidylcholine (PC) ratio decreased in the nuclear membranes, the sphingomyelin (SM) ratio increased in the microsomal membranes, and the phosphatidylethanolamine (PE) ratio increased and the phosphatidylserine (PS) ratio decreased in the mitochondrial membranes. Moreover, the mRNA expression levels of enzymes related to PC, PE, PS, PA, SM and cardiolipin syntheses changed during cell growth. We suggest that the phospholipid class compositions of organellar membranes are tightly regulated by cell growth. These findings provide a basis for future investigations of cancer cell growth and lipid metabolism

Analysis of Risk Factors for High-dose Cisplatin-induced Renal Impairment in Head and Neck Cancer Patients

Background/Aim:Concurrent chemoradiotherapy with high-dose cisplatin (CDDP-RT) is the standard therapy for advanced head and neck cancer; however, due to CDDP-induced renal impairment, dose reduction or discontinuation is frequently required. Therefore, the identification of risk factors for renal impairment is of importance to improve the efficacy and safety of CDDP-RT.Patients and Methods: We retrospectively investigated risk factors for renal impairment in advanced head and neck cancer patients receiving CDDP-RT. Renal impairment was defined as a >25% decrease from baseline in estimated glomerular filtration rate within 14 days after CDDP administration in the first cycle.Results:Of the 82 patients analyzed in this study, 21 (26%) patients developed renal impairment. Multivariate logistic regression analysis showed that concomitant use of a calcium channel blocker or lower hemoglobin levels significantly contributed to the increased risk of CDDP-induced renal impairment (odds ratio=3.60, 95% confidence interval=1.04-12.40; odds ratio=0.71, 95% confidence interval=0.50-0.99, respectively), while concomitant use of proton pump inhibitors was a factor associated with a decreased risk of CDDP-induced renal impairment (odds ratio=0.20, 95% confidence interval=0.04-0.86).Conclusion:Renal function of patients receiving calcium channel blocker or patients with lower hemoglobin levels should be monitored cautiously when receiving CDDP-RT

Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation, Contributing to Cognitive Function

BACKGROUND: Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKbeta is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We, therefore, developed DGKbeta KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKbeta. In addition, overexpression of DGKbeta in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKbeta, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKbeta but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that membrane-localized DGKbeta regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory

Urine volume to hydration volume ratio is associated with pharmacokinetics of high-dose methotrexate in patients with primary central nervous system lymphoma.

High-dose methotrexate (HD-MTX)-based chemotherapy is the first-line treatment for primary central nervous system lymphoma (PCNSL), but is associated with severe adverse effects, including myelosuppression and renal impairment. MTX is primarily excreted by the kidneys. Renal function calculated using serum creatinine (Scr) derived from muscle may be overestimated in elderly PCNSL patients. Therefore, we aimed to construct a population pharmacokinetic model in PCNSL patients and explore the factors associated with MTX clearance. Sixteen PCNSL patients (median age, 66 years) treated with HD-MTX were included, and serum MTX concentrations were measured at 193 points in 49 courses. A population pharmacokinetic analysis was performed using NONMEM. A Monte Carlo simulation was conducted, in which serum MTX concentrations were stratified into three groups of creatine clearance (Ccr) (50, 75, and 100 ml/min) with three groups of the urine volume to hydration volume (UV/HV) ratio (2). The final model was constructed as follows: MTX clearance = 4.90·(Ccr/94.5)0.456·(UV/HV)0.458. In the Monte Carlo simulation, serum MTX concentrations were below the standard values (10, 1, and 0.1 µM at 24, 48, and 72 h, respectively, after the start of the MTX administration) in most patients with UV/HV >2, even with Ccr of 50 ml/min. Conversely, half of the patients with UV/HV <1 and Ccr of 50 ml/min failed to achieve the standard values. The present results demonstrated that the UV/HV ratio was useful for describing the pharmacokinetics of MTX in PCNSL patients

Effectiveness of COVID-19 vaccination in healthcare workers in Shiga Prefecture, Japan

This study, which included serological and cellular immunity tests, evaluated whether coronavirus disease 2019 (COVID-19) vaccination adequately protected healthcare workers (HCWs) from COVID-19. Serological investigations were conducted among 1600 HCWs (mean ± standard deviation, 7.4 ± 1.4 months after the last COVID-19 vaccination). Anti-SARS-CoV-2 antibodies N-Ig, Spike-Ig (Roche), N-IgG, Spike-IgM, and -IgG (Abbott), were evaluated using a questionnaire of health condition. 161 HCWs were analyzed for cellular immunity using T-SPOT® SARS-CoV-2 kit before, and 52 HCWs were followed up until 138.3 ± 15.7 days after their third vaccination. Spike-IgG value was 954.4 ± 2282.6 AU/mL. Forty-nine of the 1600 HCWs (3.06%) had pre-existing SARS-CoV-2 infection. None of the infectious seropositive HCWs required hospitalization. T-SPOT value was 85.0 ± 84.2 SFU/106 cells before the third vaccination, which increased to 219.4 ± 230.4 SFU/106 cells immediately after, but attenuated later (to 111.1 ± 133.6 SFU/106 cells). Poor counts (< 40 SFU/106 cells) were present in 34.8% and 38.5% of HCWs before and after the third vaccination, respectively. Our findings provide insights into humoral and cellular immune responses to repeated COVID-19 vaccinations. COVID-19 vaccination was effective in protecting HCWs from serious illness during the original Wuhan-1, Alpha, Delta and also ongoing Omicron-predominance periods. However, repeated vaccinations using current vaccine versions may not induce sufficient cellular immunity in all HCWs