Identification of ß1-3 galactosylglucose-core free-glycans in human urine.

We previously reported the precise structure of acidic free-glycans in human urine. In the present study, structural analysis of neutral free-glycans in urine was performed in fine detail. Urine samples were collected from 21 healthy volunteers and free-glycans extracted from the creatinine-adjusted urine and then fluorescently labeled with 2-aminopyridine. Neutral glycan profiling was achieved by a combination of high-performance liquid chromatography, mass spectrometry, enzymatic digestion, and periodate cleavage. A total of 79 glycans were identified. Because the ABO-blood group antigen containing urinary neutral glycans are major components, profiling patterns were similar between individuals of the same ABO-group. The neutral glycans were composed of lactose-core (Galß1-4Glc) glycans, type-II N-acetyllactosamine-core (GlcNAcß1-4Glc) glycans, hexose oligomers, N-glycans and to our surprise ß1-3 galactosylglucose-core (Galß1-3Glc) glycans. Although glycans with a ß1-3 galactosylglucose-core were identified as major components in urine, comprising structurally simple isomers of a lactose-core, the core structure has not previously been reported. The major ß1-3 galactosylglucose-core glycans were Fucα1-2Galß1-3(Fucα1-4)Glc, GalNAcα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc and Galα1-3(Fucα1-2)Galß1-3(Fucα1-4)Glc, corresponding to H-, A-, and B-blood group antigens, respectively. Three lactosamine extended ß1-3 galactosylglucose-core glycans were also detected as minor components. Elucidating the biosynthesis of ß1-3 galactosylglucose will be crucial for understanding the in vivo function of these glycans.

Investigation of acidic free-glycans in urine and their alteration in cancer.

Alterations to glycans in cancer patients have been used to identify novel tumor biomarkers. Most of these studies have focused on protein glycosylation but less attention has been paid to free-glycans. Here, we analyzed acidic free-glycans in the urine of cancer patients to identify novel tumor marker candidates. Specifically, urine samples were collected from patients with gastric cancer, pancreatic cancer and cholangiocarcinoma as well as normal controls. The free-glycans were extracted from creatinine-adjusted urine and fluorescently labeled with 2-aminopyridine. Initially, we performed profiling of urinary free-glycans by high-performance liquid chromatography and mass spectrometry with enzymatic and chemical degradation. More than 100 glycans, including novel structures, were identified. The chromatographic peaks suggested some of these glycans were present at elevated levels in cancer patients. To verify cancer-associated alterations, we compared the glycan levels between cancer patients and normal controls by selected reaction monitoring. Representative structures of glycans with elevated levels in cancer patients included the following: small glycans related to sialyllactose; sialyl Lewis X; lactose- and N-acetyllactosamine (LacNAc) type-II-core glycans with LacNAc (type-I or II)-extensions and modifications of α1,3/4-fucose and/or 6-sulfate on the Glc/GlcNAc; free-N-glycans containing sialylation or ß1,6-branch of 6-sulfo Lewis X; novel NeuAcα2-3Galß1-4(+/-Fucα1-3) Xylα1-3Glc glycans. Our results provide further insight into urinary free-glycans and suggest the potential utility of these compounds as tumor markers.

Analysis of daily energy, protein, fat, and carbohydrate intake in citrin-deficient patients: Towards prevention of adult-onset type II citrullinemia.

Patients with citrin deficiency during the adaptation/compensation period exhibit diverse clinical features and have characteristic diet of high protein, high fat, and low carbohydrate. Japanese cuisine typically contains high carbohydrate but evaluation of diet of citrin-deficient patients in 2008 showed a low energy intake and a protein:fat:carbohydrate (PFC) ratio of 19:44:37, which indicates low carbohydrate consumption rate. These findings prompted the need for diet intervention to prevent the adult onset of type II citrullinemia (CTLN2). Since the publication of the report about 10 years ago, patients are generally advised to eat what they wish under active dietary consultation and intervention. In this study, citrin-deficient patients and control subjects living in the same household provided answers to a questionnaire, filled-up a maximum 6-day food diary, and supplied physical data and information on medications if any. To study the effects of the current diet, the survey collected data from 62 patients and 45 controls comparing daily intakes of energy, protein, fat, and carbohydrate. Food analysis showed that patient's energy intake was 115% compared to the Japanese standard. The confidence interval of the PFC ratio of patients was 20-22:47-51:28-32, indicating higher protein, higher fat and lower carbohydrate relative to previous reports. The mean PFC ratio of female patients (22:53:25) was significantly different from that of male patients (20:46:34), which may explain the lower frequency of CTLN2 in females. Comparison of the present data to those published 10 years ago, energy, protein, and fat intakes were significantly higher but the amount of carbohydrate consumption remained the same. Regardless of age, most patients (except for adolescents) consumed 100-200 g/day of carbohydrates, which met the estimated average requirement of 100 g/day for healthy individuals. Finally, patients were generally not overweight and some CTLN2 patients were underweight although their energy intake was higher compared with the control subjects. We speculate that high-energy of a low carbohydrate diet under dietary intervention may help citrin-deficient patients attain normal growth and prevent the onset of CTLN2.

Mental health literacy regarding eating disorders in female Japanese university students.

Mental health literacy (MHL) refers to one's knowledge and beliefs about mental disorders. MHL is necessary for the prevention and early detection of eating disorders, but little research exists on the MHL of Japanese people. This paper aims to reveal the degree of MHL about eating disorders in the Japanese individuals most susceptible to them (namely, female university students). Female Japanese university students were surveyed with a self-report questionnaire, using the Japanese versions of anorexia nervosa and bulimia nervosa vignettes. Most participants responded that the main problem described in each vignette were "eating disorders" and tended to suggest medical care for the treatment. However, many still believed these issues to be simple problems of eating behavior. Overall, the participants demonstrated inadequate MHL about eating disorders. Initiatives to improve this population's knowledge and understanding of eating disorders are necessary.

Down-regulation of Claudin-2 Expression and Proliferation by Epigenetic Inhibitors in Human Lung Adenocarcinoma A549 Cells.

Claudin-2 is highly expressed in lung adenocarcinoma tissues and increases proliferation in adenocarcinoma cells. The chemicals that reduce claudin-2 expression may have anti-cancer effects, but such therapeutic medicines have not been developed. We found that azacitidine (AZA), a DNA methylation inhibitor, and trichostatin A (TSA) and sodium butyrate (NaB), histone deacetylase (HDAC) inhibitors, decrease claudin-2 levels. The effect of AZA was mediated by the inhibition of phosphorylated Akt and NF-κB. LY-294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and BAY 11-7082, an NF-κB inhibitor, decreased claudin-2 levels. The reporter activity of claudin-2 was decreased by AZA and LY-294002, which was blocked by the mutation in a putative NF-κB-binding site. NF-κB bound to the promoter region of claudin-2, which was inhibited by AZA and LY-294002. AZA is suggested to decrease the claudin-2 mRNA level mediated by the inhibition of a PI3K/Akt/NF-κB pathway. TSA and NaB did not change phosphorylated Akt and NF-κB levels. Furthermore, these inhibitors did not change the reporter activity of claudin-2 but decreased the stability of claudin-2 mRNA mediated by the elevation of miR-497 microRNA. The binding of histone H3 to the promoter region of miR-497 was inhibited by TSA and NaB, whereas that of claudin-2 was not. These results suggest that HDAC inhibitors decrease claudin-2 levels mediated by the elevation of miR-497 expression. Cell proliferation was additively decreased by AZA, TSA, and NaB, which was partially rescued by ectopic expression of claudin-2. We suggest that epigenetic inhibitors suppress the abnormal proliferation of lung adenocarcinoma cells highly expressing claudin-2.

Identification of internally sialylated carbohydrate tumor marker candidates, including Sda/CAD antigens, by focused glycomic analyses utilizing the substrate specificity of neuraminidase.

In our previous study, 14 sulfated carbohydrate tumor marker candidates were identified by focused glycomic analyses. Here, glycomic analyses focused on internally sialylated glycans to identify novel marker candidates. Internally sialylated glycans were enriched by digestion of pyridylaminated glycans prepared from sera with α-neuraminidase from Salmonella typhimurium, which did not cleave sialic acids linked to internal residues, followed by anion-exchange chromatography. Next, internally sialylated O-glycan profiles were constructed using two types of high performance liquid chromatography, which were compared between 20 healthy controls and 11 patients with gastric cancer and 9 patients with pancreatic cancer. In all, 17 marker candidates were identified. The structures of glycan candidates were precisely analyzed using enzymatic digestion, glycan synthesis, 2D mapping and mass spectrometry. Among 17 candidates, one was STn, and the other 16 comprised 10 core1, 1 core2 and 5 core3 glycans. The various structures included a α2,6-sialylated reducing terminal GalNAc and α2,6-sialylated type1 N-acetyl-lactosamine. Eight candidates possessed the Sda/CAD antigen. The levels of these candidate glycans in sera from all 40 subjects were quantified using a selected reaction monitoring assay and found to be elevated in at least one or more patients. Although the serum levels of each candidate glycan varied between patients, those candidates having the same backbone or determinant, such as core3 backbone and core1 structures with extended type1 N-acetyl-lactosamine, displayed similar patterns of elevation. These results suggest that analysis of multiple markers may be an effective means of diagnosing various cancers.

Correlation of serum sialyl Tn antigen values determined by immunoassay and SRM based method.

We previously identified four glycan tumor marker candidates using a HPLC-based method. One candidate was sialyl Tn (STN), NeuAcα2-6-GalNAc. In this study, glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine. Pyridylaminated-STN levels of 147 gastric cancer, 85 pancreatic cancer and 10 cholangiocarcinoma patients together with 102 normal controls were accurately quantified using HPLC separation followed by selected reaction monitoring (SRM) assay, which used a stable isotope, tetradeuterium-labeled pyridylamino glycan as an internal standard. Additionally, STN values were also quantified using conventional competitive inhibition radioimmunoassay (RIA). The two STN levels determined by RIA and SRM gave a similar distribution pattern in sera. STN levels were increased in sera from cancer patients compared to those from normal controls. Moreover, the STN levels in sera of cancer patients determined by the two different assay procedures showed a good correlation (i.e., correlation coefficient >0.9). Our results suggest it may be better to determine STN levels using SRM instead of RIA.

Various sulfated carbohydrate tumor marker candidates identified by focused glycomic analyses.

Glycomic analysis focused on sulfated O-glycans was performed to identify novel serum carbohydrate tumor markers. Sulfated glycans were enriched by α-neuraminidase digestion of pyridylaminated glycans prepared from sera, followed by anion exchange chromatography. Sulfated O-glycan profiles were constructed by two types of high performance liquid chromatography separation. Comparison of the profiles from 20 healthy controls with those of 11 gastric and 9 pancreatic cancer patients identified 14 marker candidates. The structures of these candidates were precisely analyzed using various methods including enzymatic digestion and mass spectrometry. The candidates comprised 9 core1 and 5 core2 glycans. All these candidates were monosulfated, and 11 were also mono- or difucosylated, and included various determinants such as 6-sulfo type2 lactosamine, 6-sulfo Lewis X, 6-sulfo Lewis Y, 3'-sulfo type1 lactosamine and 3'-sulfo Lewis A. Furthermore, among the core1 glycans, five candidates displayed a type1 and type2 lactosamine hybrid backbone. The levels of these candidate glycans in the sera from all 40 subjects were quantified using a selected reaction monitoring assay. These analyses revealed: (i) the levels of all candidates were elevated in sera of at least one or more patients; (ii) core1 candidates having type1-type2 hybrid backbones with 6-sulfo Lewis X, 6-sulfo type2 lactosamine or 3'-sulfo Lewis A were elevated in sera of variety of patients; and (iii) levels of the candidates varied widely among patients, suggesting analysis of multiple candidates will be an effective means of screening various cancers. To fully evaluate the clinical utility of these candidates, a further verification study is required.

Gravitropism interferes with hydrotropism via counteracting auxin dynamics in cucumber roots: clinorotation and spaceflight experiments.

Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (µG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under µG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and µG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in µG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.

Elevation of CA19-9-Related Novel Marker, Core 1 Sialyl Lewis A, in Sera of Adenocarcinoma Patients Verified by a SRM-Based Method.

We have attempted to identify a novel glycan tumor marker. Pyridylaminated (PA) O-glycans were prepared from sera, and the corresponding O-glycan profiles were constructed by HPLC separation. By comparing the serum O-glycan profiles from healthy controls with those of cancer patients, we identified a marker candidate, core 1 sialyl Lewis A (NeuAcα2-3Galß1-3(Fucα1-4)GlcNAcß1-3Gal) (abbreviated C1SLA), whose concentration appeared to be weakly correlated with CA19-9 values. To quantify this glycan, we developed a selected reaction monitoring (SRM) assay that used a stable isotope, tetradeuterium-labeled pyridylamino (d4-PA) glycan, as an internal standard. The analyte (d0-PA-C1SLA) and the internal standard (d4-PA-C1SLA) were subjected to SRM analyses after two types of HPLC separation. Serum levels of C1SLA, determined as the relative ratio to total O-glycans, were then measured. These analyses revealed that (i) C1SLA is a CA19-9-related glycan, (ii) the mean value of C1SLA in normal controls is 3.41 ppm, (iii) the level of C1SLA was significantly higher in samples of stages II-IV stomach cancers (P = 0.0036) as well as pancreatic cancers (P < 0.0001) compared to that of normal controls, (iv) the relationship between C1SLA and CA19-9 varies from poor to weak depending on the cancer, and (v) C1SLA could be valuable as a diagnostic adjunct for cancer.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus.

Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

Deletion of the presynaptic scaffold CAST reduces active zone size in rod photoreceptors and impairs visual processing.

How size and shape of presynaptic active zones are regulated at the molecular level has remained elusive. Here we provide insight from studying rod photoreceptor ribbon-type active zones after disruption of CAST/ERC2, one of the cytomatrix of the active zone (CAZ) proteins. Rod photoreceptors were present in normal numbers, and the a-wave of the electroretinogram (ERG)--reflecting their physiological population response--was unchanged in CAST knock-out (CAST(-/-)) mice. Using immunofluorescence and electron microscopy, we found that the size of the rod presynaptic active zones, their Ca(2+) channel complement, and the extension of the outer plexiform layer were diminished. Moreover, we observed sprouting of horizontal and bipolar cells toward the outer nuclear layer indicating impaired rod transmitter release. However, rod synapses of CAST(-/-) mice, unlike in mouse mutants for the CAZ protein Bassoon, displayed anchored ribbons, normal vesicle densities, clustered Ca(2+) channels, and essentially normal molecular organization. The reduction of the rod active zone size went along with diminished amplitudes of the b-wave in scotopic ERGs. Assuming, based on the otherwise intact synaptic structure, an unaltered function of the remaining release apparatus, we take our finding to suggest a scaling of release rate with the size of the active zone. Multielectrode-array recordings of retinal ganglion cells showed decreased contrast sensitivity. This was also observed by optometry, which, moreover, revealed reduced visual acuity. We conclude that CAST supports large active zone size and high rates of transmission at rod ribbon synapses, which are required for normal vision.

Involvement of afadin in barrier function and homeostasis of mouse intestinal epithelia.

Afadin interacts with the cytoplasmic region of nectins, which are immunoglobulin-like cell adhesion molecules at adherens junctions, and links them to the actin cytoskeleton. Afadin regulates activities of cells in culture such as directional motility, proliferation and survival. We used Cre-loxP technology to generate mice conditionally lacking afadin specifically in the intestinal epithelia after birth. The loss of afadin caused increased paracellular permeability in the intestinal mucosa and enhanced susceptibility to the tissue destruction induced by dextran sulfate sodium. The junctional architecture of the intestinal epithelia appeared to be preserved, whereas the deficiency of afadin caused the mislocalization of nectin-2 and nectin-3 from adherens junctions to basolateral membrane domains but not that of other components of apical junctions. By contrast, such phenotypic changes were undetected in mice lacking nectin-2, nectin-3 or both. These findings suggest that afadin plays crucial roles, independently of the role as the nectin-afadin module, in barrier function and homeostasis of the intestinal epithelia once the epithelial structure has been established.

Occurrence of a D-arabinose-containing complex-type free-N-glycan in the urine of cancer patients.

Urinary free-glycans are promising markers of disease. In this study, we attempted to identify novel tumor markers by focusing on neutral free-glycans in urine. Free-glycans extracted from the urine of normal subjects and cancer patients with gastric, colorectal, pancreatic and bile duct were fluorescently labeled with 2-aminopyridine. Profiles of these neutral free-glycans constructed using multidimensional high performance liquid chromatography separation were compared between normal controls and cancer patients. The analysis identified one glycan in the urine of cancer patients with a unique structure, which included a pentose residue. To reveal the glycan structure, the linkage fashion, monosaccharide species and enantiomer of the pentose were analyzed by high performance liquid chromatography and mass spectrometry combined with several chemical treatments. The backbone of the glycan was a monoantennary complex-type free-N-glycan containing ß1,4-branch. The pentose residue was attached to the antennal GlcNAc and released by α1,3/4-L-fucosidase. Intriguingly, the pentose residue was consistent with D-arabinose. Collectively, this glycan structure was determined to be Galß1-4(D-Araß1-3)GlcNAcß1-4Manα1-3Manß1-4GlcNAc-PA. Elevation of D-arabinose-containing free-glycans in the urine of cancer patients was confirmed by selected reaction monitoring. This is the first study to unequivocally show the occurrence of a D-arabinose-containing oligosaccharide in human together with its detailed structure.

Increased levels of acidic free-N-glycans, including multi-antennary and fucosylated structures, in the urine of cancer patients.

We recently reported increased levels of urinary free-glycans in some cancer patients. Here, we focused on cancer related alterations in the levels of high molecular weight free-glycans. The rationale for this study was that branching, elongation, fucosylation and sialylation, which lead to increases in the molecular weight of glycans, are known to be up-regulated in cancer. Urine samples from patients with gastric cancer, pancreatic cancer, cholangiocarcinoma and colorectal cancer and normal controls were analyzed. The extracted free-glycans were fluorescently labeled with 2-aminopyridine and analyzed by multi-step liquid chromatography. Comparison of the glycan profiles revealed increased levels of glycans in some cancer patients. Structural analysis of the glycans was carried out by performing chromatography and mass spectrometry together with enzymatic or chemical treatments. To compare glycan levels between samples with high sensitivity and selectivity, simultaneous measurements by reversed-phase liquid chromatography-selected ion monitoring of mass spectrometry were also performed. As a result, three lactose-core glycans and 78 free-N-glycans (one phosphorylated oligomannose-type, four sialylated hybrid-type and 73 bi-, tri- and tetra-antennary complex-type structures) were identified. Among them, glycans with α1,3-fucosylation ((+/- sialyl) Lewis X), triply α2,6-sialylated tri-antennary structures and/or a (Man3)GlcNAc1-core displayed elevated levels in cancer patients. However, simple α2,3-sialylation and α1,6-core-fucosylation did not appear to contribute to the observed increase in the level of glycans. Interestingly, one tri-antennary free-N-glycan that showed remarkable elevation in some cancer patients contained a unique Glcß1-4GlcNAc-core instead of the common GlcNAc2-core at the reducing end. This study provides further insights into free-glycans as potential tumor markers and their processing pathways in cancer.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Vascular smooth muscle RhoA counteracts abdominal aortic aneurysm formation by modulating MAP4K4 activity.

Whether a small GTPase RhoA plays a role in the pathology of abdominal aortic aneurysm (AAA) has not been determined. We show here that RhoA expression is reduced in human AAA lesions, compared with normal areas. Furthermore, incidence of AAA formation is increased in vascular smooth muscle cell (VSMC)-specific RhoA conditional knockout (cKO) mice. The contractility of the aortic rings and VSMCs from RhoA cKO mice is reduced, and expression of genes related to the VSMC contractility is attenuated by loss of RhoA. RhoA depletion activates the mitogen-activated protein (MAP) kinase signaling, including MAP4K4, in the aorta and VSMCs. Inhibition of MAP4K4 activity by DMX-5804 decreases AAA formation. Set, a binding protein to active RhoA, functions as an activator of MAP4K4 by sequestering PP2A, an inhibitor of MAP4K4, in the absence of RhoA. In conclusion, RhoA counteracts AAA formation through inhibition of MAP4K4 in cooperation with Set.

Food Preferences of Patients with Citrin Deficiency.

Citrin deficiency is characterized by a wide range of symptoms from infancy through adulthood and presents a distinct preference for a diet composed of high protein, high fat, and low carbohydrate. The present study elucidates the important criteria by patients with citrin deficiency for food selection through detailed analysis of their food preferences. The survey was conducted in 70 citrin-deficient patients aged 2-63 years and 55 control subjects aged 2-74 years and inquired about their preference for 435 food items using a scale of 1-4 (the higher, the more favored). The results showed that the foods marked as "dislike" accounted for 36.5% in the patient group, significantly higher than the 16.0% in the controls. The results also showed that patients clearly disliked foods with 20-24 (% of energy) or less protein, 45-54% (of energy) or less fat, and 30-39% (of energy) or more carbohydrate. Multiple regression analysis showed carbohydrates had the strongest influence on patients' food preference (ß = -0.503). It also showed female patients had a stronger aversion to foods with high carbohydrates than males. The protein, fat, and carbohydrate energy ratio (PFC) of highly favored foods among patients was almost the same as the average PFC ratio of their daily diet (protein 20-22: fat 47-51: carbohydrates 28-32). The data strongly suggest that from early infancy, patients start aspiring to a nutritional balance that can compensate for the metabolism dissonance caused by citrin deficiency in every food.

Dynamic regulation of p53 subnuclear localization and senescence by MORC3.

The tumor suppressor p53 is a key transcriptional factor regulating the induction of cellular senescence by oncogenic signals. The activity of p53 is regulated by recruitment into promyelocytic leukemia (PML)-nuclear bodies (NBs) as well as by stabilization through posttranslational modifications such as phosphorylation and acetylation. Here we found that MORC3 (microrchidia3)-ATPase activated p53 and induced cellular senescence in normal human and mouse fibroblasts but not p53-/- fibroblasts. Conversely, genotoxic stress-induced phosphorylation and stabilization of p53 but barely increased its transcriptional activity in Morc3-/- fibroblasts. MORC3 localized on PML-NBs in presence of PML and mediated recruitment of p53 and CREB-binding protein (CBP) into PML-NBs. In contrast, expression of ATPase activity-deficient mutant MORC3-E35A or siRNA repression of MORC3 impaired the localization of p53 and Sp100 but not CBP on PML-NBs. These results suggest that MORC3 regulates p53 activity and localization into PML-NBs. We identified a new molecular mechanism that regulates the activity of nuclear proteins by localization to a nuclear subdomain.

Increased susceptibility to spontaneous lung cancer in mice lacking LIM-domain only 7.

LIM-domain only (LMO) 7 is a multifunctional protein that is predicted to regulate the actin cytoskeleton, assembly of adherens junctions in epithelial cells, and gene expression. LMO7 was highly expressed in the mouse lung and predominantly localized to the apical membrane domain of bronchiolar epithelial cells. Although mice lacking LMO7 were viable and fertile in specific pathogen-free conditions, they developed protruding epithelial lesions in the terminal and respiratory bronchioles and alveolar ducts at 14-15 weeks of age. Furthermore, they tended to develop spontaneous adenocarcinoma in the lung at over 90 weeks of age. The cumulative incidence ratios of lung cancer were 22% in LMO7(-/-) mice and 13% in LMO7(+/-) mice whereas no primary lung cancer was observed in wild-type mice. Ex vivo analyses of the cancer cells showed numerical chromosome abnormalities and tumorigenicity in nude mice. These results suggest that LMO7 can act as a tumor suppressor whose deficiency confers a genetic predisposition to naturally occurring lung cancer.