Greater consumption of noodle is associated with higher serum phosphorus levels: a cross-sectional study on healthy participants.

Higher serum phosphorus levels are associated with mortality and cardiovascular events, both in healthy individuals and those with chronic kidney disease. Owing to the increasing westernization of eating habits, a decrease in rice consumption and an increase in the intake of bread and noodle products were observed in Japan. This cross-sectional study investigated the influence of staple food (rice, bread, and noodle) consumption patterns on the serum levels of phosphorus and phosphate-regulating factors in 103 healthy young participants. Fasting blood and 24-h urine samples were collected; data about dietary habits were collected using a brief-type self-administered diet history questionnaire. Cluster analysis was conducted to assess subgroups classified according to staple food consumption patterns. Serum levels of phosphorus and phosphate-regulating factors did not significantly differ between subgroups classified based on the frequency of rice or bread consumption. However, the serum levels of phosphorus and fibroblast growth factor 23 were more significantly elevated in the higher than in the lower noodle consumption frequency subgroup. Cluster analysis defined three clusters, and the serum phosphorus levels in the high-noodle cluster were significantly higher than that in the high-bread and high-rice clusters. A high consumption of noodles was associated with elevated serum phosphorus levels. The trial was registered in the University Hospital Medical Information Network (UMIN) Center system (The name of the trial register: Hidekazu Arai, and UMIN accession number: UMIN000034352).

Collective cancer cell invasion in contact with fibroblasts through integrin-α5ß1/fibronectin interaction in collagen matrix.

Interaction of cancer cells with cancer-associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3-D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin-α5ß1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E-cadherin-expressing adenocarcinoma cell lines, DLD-1 (colon) and MCF-7 (breast). When hybrid spheroids of DLD-1 cells with CAFs were embedded into collagen gel, DLD-1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor-α promoted the collective invasion, possibly by reducing the E-cadherin junction, as did the transforming growth factor-ß inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor-ß itself inhibited the cancer cell invasion. Efficient collective invasion of DLD-1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function-blocking Abs and siRNAs confirmed that DLD-1 cells adhered to fibronectin fibrils on CAFs mainly through integrin-α5ß1. Anti-E-cadherin Ab promoted the single cell invasion of DLD-1 cells by dissociating the E-cadherin junction. Although the binding affinity of MCF-7 cells to CAFs was lower than DLD-1, they also collectively invaded the collagen matrix in a similar fashion to DLD-1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.

Carbohydrate antigen 19-9 is significantly elevated in autosomal dominant polycystic kidney disease.

AIM: Liver cysts are the most common extrarenal manifestation in patients with autosomal dominant polycystic kidney disease (ADPKD). Carbohydrate antigen 19-9 (CA19-9) is generally used as a marker for biliopancreatic malignancies, although CA19-9 levels in patients with ADPKD are largely unknown. METHODS: A prospective observational study of 53 ADPKD patients and 83 non-ADPKD control subjects was performed. The serum levels of CA19-9 were studied to evaluate the association with clinical parameters and liver cysts. RESULTS: The serum CA19-9 levels were significantly higher in the ADPKD group than in the control group (32.9 U/mL vs. 9.8 U/mL, respectively, P < 0.001). The serum CA19-9 levels in the ADPKD group were positively correlated with the mean blood pressure (rho = 0.335, P < 0.05), gamma-glutamyl transferase (GTP) levels (rho = 0.541, P < 0.001), the largest cyst size (rho = 0.536, P < 0.001) and the liver cyst volume (rho = 0.682, P < 0.001). Multiple regression analyses showed that the gamma-GTP levels (P < 0.001) and the liver cyst volumes (P < 0.001) were independent predictors for serum CA19-9 levels. CONCLUSIONS: Serum CA19-9 levels are significantly elevated and appear to be dependent on the gamma-GTP levels and the volume of liver cysts in patients with ADPKD. Our findings indicate that the measurement of the baseline CA19-9 level in each patient with ADPKD may be useful for the interpretation of the value and the differential diagnosis of liver diseases, particularly the liver cyst infection.

Effect of endurance training and branched-chain amino acids on the signaling for muscle protein synthesis in CKD model rats fed a low-protein diet.

A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched-chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately one-half of the animals in each group was subjected to treadmill exercise (15 m/min, 1 h/day, 5 days/wk). After 7 wk, renal function was measured, and soleus muscles were collected to evaluate muscle protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified.

Quantitative assessment of locomotive syndrome by the loco-check questionnaire in older Japanese females.

[Purpose] Locomotive syndrome (LS) is a condition by which older people may require care service because of problems with locomotive organs. This study examined whether the loco-check, a 7-item questionnaire, is useful for quantitatively assessing the severity of LS. [Subjects and Methods] Seventy-one community dwelling Japanese females aged 64-96 years (81.7 ± 8.0 years) participated in this study. The associations of the loco-check with thigh muscle mass measured by X-ray CT, physical performance, nutritional status, and quality of life (QOL) were investigated. [Results] The results showed that the number of times that "yes" was selected in the loco-check was significantly correlated with thigh muscle mass, major measures of physical performance, nutritional status, and QOL. This number was also significantly larger in the participants experiencing falling, fracture, and lumbar pain than in those without these episodes. [Conclusion] These results suggest that the loco-check might be useful for quantitatively evaluating LS.

[Chronic kidney disease and nutrition].

Abnormalities of mineral metabolism develop with decline of renal function in chronic kidney disease (CKD), and it is called as a CKD-mineral and bone disorder (CKD-MBD). The standard approach for management of CKD-MBD is to keep serum phosphorus, calcium, and parathyroid hormone in the reference range by dietary intervention and medications. It has been recently pointed out that starting the treatment from early CKD is important for suppressing CKD-MBD.

Impact of hyperuricaemia on the chronic kidney disease-associated risk factors in a community-based population.

AIM: Hyperuricaemia is a common finding in subjects with lifestyle related diseases. This study was performed to examine its association with chronic kidney disease (CKD) in relation to other risk factors in a community-based population. METHODS: Data from 187 914 participants, excepting CKD stage 5, of the health check-up were included in this analysis. The association between CKD and its risk factors were examined by a logistic analysis. The association of hyperuricaemia and CKD was also compared in the population without any lifestyle related diseases and the whole population. RESULTS: The prevalence of hyperuricaemia was significantly higher in the advanced stage of CKD. The odds ratio of hyperuricaemia was higher than that of other factors for the association with CKD. The odds ratio of many CKD-associated variables was increased in the advanced stage. Among them, the odds ratio of hyperuricaemia was markedly increased. The prevalence of hyperuricaemia was lower in the population without any lifestyle related diseases than in the whole population in the early stages, the difference of prevalence between the two populations becoming smaller in the advanced stage. CONCLUSIONS: The association of incident CKD with hyperuricaemia was stronger than with other chronic kidney disease-risk factors, this association becoming more significant in the advanced stage of chronic kidney disease. Although this result does not indicate the cause and result relationship, the data suggest that hyperuricaemia might not be appropriately treated in this population.

Characterization of genome-reduced fission yeast strains.

The Schizosaccharomyces pombe genome is one of the smallest among the free-living eukaryotes. We further reduced the S. pombe gene number by large-scale gene deletion to identify a minimal gene set required for growth under laboratory conditions. The genome-reduced strain has four deletion regions: 168.4 kb in the left arm of chromosome I, 155.4 kb in the right arm of chromosome I, 211.7 kb in the left arm of chromosome II and 121.6 kb in the right arm of chromosome II. The deletions corresponded to a loss of 223 genes of the original ~5100. The quadruple-deletion strain, with a total deletion size of 657.3 kb, showed a decreased ability to uptake glucose and some amino acids in comparison with the parental strain. The strain also showed increased gene expression of the mating pheromone M-factor precursor and the nicotinamide adenine dinucleotide phosphate -specific glutamate dehydrogenase. There was also a 2.7-fold increase in the concentration of cellular adenosine triphosphate, and levels of the heterologous proteins, enhanced green fluorescent protein and secreted human growth hormone were increased by 1.7- and 1.8-fold, respectively. The transcriptome data from this study have been submitted to the Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE38620 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vjkxjewuywgcovc&acc=GSE38620).

Protective effects of relaxin against cisplatin-induced nephrotoxicity in rats.

BACKGROUND: Cisplatin (CDDP)-induced acute kidney injury (AKI) involves pro-inflammatory responses, apoptosis of renal tubular epithelial cells and vascular damage. AKI increases the risk of chronic kidney disease. Relaxin (RLX) has anti-apoptotic and anti-fibrosis properties. The aim of this study was to investigate the effects of RLX on CDDP-induced nephrotoxicity. METHODS: We investigated the mitigating effects of RLX based on the etiopathology of AKI induced by CDDP, and also the anti-fibrotic effect of RLX on renal fibrosis after AKI. In the short-term experiments, rats were divided into the control group, CDDP group, and CDDP+RLX group. In the latter group, RLX was infused for 5 or 14 days using an implanted osmotic minipump. CDDP was injected intraperitoneally (6 mg/kg) after RLX or saline infusion. At 5 and 14 days post-CDDP, the kidneys were removed for analysis. The effect of RLX on renal fibrosis after AKI was evaluated at 6 weeks post-CDDP. RESULTS: In short-term experiments, CDDP transiently increased plasma creatinine and blood urea nitrogen with peaks at day 5, and RLX prevented such rises. Semiquantitative analysis of the histological lesions indicated marked structural damage and apoptotic cells in the CDDP group, with the lesions being reduced by RLX treatment. Overexpression of Bax, interleukin-6 and tumor necrosis factor-α observed in the kidneys of the CDDP group was reduced in the CDDP+RLX group. In the long-term experiments, RLX significantly reduced renal fibrosis compared with the CDDP group. CONCLUSIONS: The results suggested that RLX provided protection against CDDP-induced AKI and subsequent fibrosis by reducing apoptosis and inflammation.

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.

Plasma levels of the pro-inflammatory protein S100A12 (EN-RAGE) are associated with muscle and fat mass in hemodialysis patients: a cross-sectional study.

BACKGROUND: Malnutrition is highly prevalent and contributes to mortality in hemodialysis (HD) patients. Although the receptor for advanced glycation end products (RAGE) system also contributes to the morbidity and mortality of these patients, the role that the RAGE system plays in determining nutritional status is currently unknown. METHODS: A cross-sectional study examining 79 HD patients was performed. The plasma concentrations of the soluble RAGE (sRAGE) and S100A12 (also known as EN-RAGE) were studied to evaluate their association with nutritional status, which was assessed by measuring the mid-thigh muscle mass and subcutaneous fat mass with computed tomography. RESULTS: Plasma S100A12 concentrations were shown to be significantly and negatively correlated with muscle mass and with fat mass (r = -0.237, P < 0.05 and r = -0.261, P < 0.05, respectively). In contrast, sRAGE was not shown to significantly correlate with either of these factors. Multiple regression analyses demonstrated that S100A12 is a significant independent predictor of both muscle mass and fat mass (P < 0.01 and P < 0.05, respectively). CONCLUSIONS: Our findings suggest that plasma S100A12 levels could play an important role in determining muscle mass and fat mass in HD patients. TRIAL REGISTRATION: Study number; UMIN000012341.

Plasma levels of fibroblast growth factor-23 are associated with muscle mass in haemodialysis patients.

AIM: Malnutrition is highly prevalent in haemodialysis (HD) patients, and it contributes to morbidity and mortality. Fibroblast growth factor-23 (FGF-23) and Klotho contribute to chronic kidney disease-mineral and bone disorder (CKD-MBD) in HD patients, but the role that these molecules play in determining nutritional status is currently unknown. METHODS: A cross-sectional study examining 77 HD patients was performed. The plasma concentrations of FGF-23 and soluble Klotho (s-Klotho) were studied to evaluate their association with muscle mass, which was investigated by abdominal muscle areas measured using computed tomography and by creatinine (Cr) production estimated using the Cr kinetic model. RESULTS: Plasma FGF-23 concentrations were significantly and positively correlated with abdominal muscle areas and Cr production (rho = 0.301, P < 0.01 and rho = 0.345, P < 0.01, respectively). In contrast, s-Klotho was not significantly correlated with these muscle mass indices and plasma FGF-23 concentrations. Multiple regression analyses showed that FGF-23 was a significant independent predictor of both muscle mass indices (P < 0.01 and P < 0.05, respectively). CONCLUSION: Plasma FGF-23 concentrations were associated with muscle mass indices in HD patients. Our findings suggest that FGF-23 and nutritional status are linked and this link is most likely independent of s-Klotho.

Relaxin protects against renal ischemia-reperfusion injury.

Relaxin, a pregnancy hormone, has antiapoptotic and anti-inflammatory properties. The aim of this study was to determine the effects of relaxin on ischemia-reperfusion (IR)-induced acute kidney injury. Male rats underwent unilateral nephrectomy and contralateral renal IR (45 min of renal pedicle clamping). Rats were divided into three groups: 1) sham group, 2) IR group, and 3) IR-RLX group (rats treated with relaxin before ischemia). In this group, relaxin was infused at 500 ng/h via subcutaneous osmotic minipump for 24 h beginning 2 h before renal ischemia. At 24 h after reperfusion, renal function was assessed and kidneys were removed for analysis. There was no significant difference in blood pressure among the three groups. IR increased plasma levels of creatinine and urea nitrogen, and relaxin provided protection against the increases in these two parameters. Relaxin significantly decreased plasma TNF-α levels and renal TNF receptor 1 mRNA expression, compared with the IR group. Semiquantitative assessment of the histological lesions showed marked structural damage in IR rats compared with the IR-RLX rats. RLX significantly reduced apoptotic cell counts compared with the IR group. Overexpression of caspase-3 observed in the IR kidneys was reduced in the IR-RLX group. The results demonstrated that relaxin provided protection against IR-induced renal injury by reducing apoptosis and inflammation.

Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe.

We have succeeded to produce a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we solved the problem by identifying plg7, encoding phospholipase A2, as a multicopy suppressor. Characterization of the RA-tolerant strains suggested that the removal of RA moieties from phospholipids would be the suppression mechanism by plg7. In this study, we extended our analysis and report our new discovery that the overexpression of plg7 enabled cells to secrete free RA into culture media. When the FAH12 integrant in the absence of the overexpressed plg7 was grown at 20 °C for 11 days, the amount of intracellular RA reached 200.1 µg/ml of culture and only 69.3 µg/ml of RA was detected in culture media. On the other hand, the FAH12 integrant harboring the plg7 multicopy plasmid secreted RA in the media (184.5 µg/ml) without decreasing the amount in the cells, i.e., a significantly higher total secretion and a lead to making RA by its secretory production in S. pombe.

Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog.

In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30 °C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.

Inflammatory factors for hypoalbuminemia in Japanese peritoneal dialysis patients.

AIM: Hypoalbuminaemia is a common complication of peritoneal dialysis (PD), and the leakage of albumin through peritoneal membrane may be a principal reason for hypoalbuminaemia. However, the relationship between peritoneal inflammation, peritoneal transport properties and hypoalbuminaemia has not been fully elucidated. METHODS: A cross-sectional study was performed on 76 Japanese PD patients who had been using a low-glucose PD solution and icodextrin. Systemic inflammatory markers of C-reactive protein (CRP) and serum interleukin-6 (IL-6), peritoneal effluent markers of dialysate IL-6 and CA125, the dialysate-to-plasma ratio of creatinine (D/Pcr) and the dialysate protein concentration were measured and examined for their relationship with hypoalbuminaemia. RESULTS: There was a significant positive correlation between serum IL-6 and dialysate IL-6, mean dialysate IL-6 being significantly higher than mean serum IL-6, suggesting that intraperitoneal inflammation was a principal origin of systemic inflammation. Both serum and dialysate IL-6 were significantly correlated with serum albumin (r= -0.25, P<0.05 and r=-0.32, P<0.01, respectively). Dialysate IL-6 was significantly correlated with D/Pcr and the dialysate protein concentration, and there was a significantly positive association between D/Pcr and the dialysate protein concentration. Dialysate CA125, which is argued to be a marker of mesothelial cell mass in this study, was positively correlated with D/Pcr and the dialysate protein concentration. The dialysate protein, dialysate IL-6 and dialysate CA125 all increased according to the peritoneal transport rate defined by D/Pcr. A multiple-regression analysis showed that serum albumin was independently associated with the age, D/Pcr and serum IL-6. CONCLUSION: Hypoalbuminaemia was attributable to both the increased peritoneal permeability and systemic inflammation, and intraperitoneal inflammation might contribute to developing these complications.

Cauterization of Narrow Root Canals Untouched by Instruments by High-Frequency Current.

The mechanical removal of bacteria is fundamental to the treatment of infected root canals, but complete sterilization of biofilms tends not to extend to uninstrumented areas. However, during electrical conduction to a root canal filled with a conductor, the higher impedance where the root canal is narrower generates Joule heat that may result in a large temperature increase and sterilization. The effect of a high-frequency electric current on the wall of a simulated narrow root canal was investigated by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). Simulated root canals, 0.1 mm in diameter, were prepared in dentine blocks. The root canal wall was treated with Plank-Rychlo solution for 5 min to create a decalcified layer. The simulated root canal was filled with either saline or NaClO, and 150 or 225 V at 520 kHz was applied for 0 s, 1 s, or 5 s. As the conduction time increased, and when the saline was replaced with NaClO, the proportion with a flat decalcified surface decreased, dentinal tubules and a lava-like morphology were significantly more evident on SEM (p < 0.01), and EDS showed significant decreases in carbon and oxygen and increases in calcium (p < 0.01). It was concluded that filling uninstrumented root canals with NaClO and using electrical conduction for 5 s could incinerate and eliminate the organic material of the root canal wall. The application of high-frequency electric current may lead to the cure of many cases of persistent apical periodontitis.

Relaxin ameliorates salt-sensitive hypertension and renal fibrosis.

BACKGROUND: Although relaxin (RLX) has potent vasodilatory and anti-fibrotic properties, there is no information on its effects on salt-sensitive hypertension. METHODS: We investigated the effects of short-term treatment with RLX on blood pressure (BP) and nitric oxide synthase (NOS) protein in the kidneys of male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats after 1 week consumption of an 8% NaCl diet. We also evaluated the inhibitory effects of each specific NOS inhibitor on BP during 1-week RLX treatment under high-salt diet. Next, we examined the long-term effects of RLX treatment for 6 weeks on renal histology and transforming growth factor-beta1 (TGF-ß1) expression in male DS and DR rats placed on the 8-week high-salt diet. RESULTS: The short-term RLX treatment significantly attenuated the high-salt diet-induced rise in BP in DS rats with increasing neuronal NOS and endothelial NOS protein in kidneys. Selective inhibition of each of the three NOS isoforms significantly blocked the anti-hypertensive effects of RLX in DS rats after 1-week high-salt diet. The long-term treatment of DS rats with RLX for 6 weeks significantly reduced systolic BP, lessened glomerular and tubulointerstitial changes and reduced TGF-ß signaling compared to saline-treated controls. CONCLUSIONS: The results suggested that RLX converted salt sensitivity to salt resistance, at least in part, by up-regulating NOS. RLX is a potentially useful therapeutic agent for salt-sensitive hypertension.

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe.

In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 µg/ml of culture that corresponded to 52.6% of total FA.

Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe.

Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.