Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus).

Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5- 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2- 3.1 and 0.9- 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised ß-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).

Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Compositional analysis of chondroitin/dermatan sulfate in rhesus monkeys (Macaca mulatta).

Glycosaminoglycans (GAGs) are found in various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of this study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A and C types. The E and iE types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

Comparison of Japanese Centenarians' and Noncentenarians' Medical Expenditures in the Last Year of Life.

Importance: Although research has shown that centenarians tend to experience shorter periods of serious illness compared with other age groups, few studies have focused on the medical expenditures of centenarians as a potential indicator of the scale of medical resources used in their last year of life. Objective: To compare Japanese centenarians' and noncentenarians' monthly medical expenditures during the year before death according to age and sex. Design, Setting, and Participants: This retrospective cohort study used linked national health and long-term care insurance data collected from April 2013 to March 2018 in Nara Prefecture, Japan, for residents aged 75 years or older who were insured under the Medical Care System for older adults and died between April 2014 and March 2018. Data were analyzed from April 2013 to March 2018. Exposures: Age of 100 years or older (centenarians) vs 75 to 99 years (noncentenarians). Main Outcomes and Measures: The numbers of unique inpatients and outpatients and medical expenditures related to decedents' hospitalization and outpatient care were extracted and analyzed based on sex and age group. The Jonckheere-Terpstra test was used to identify trends in unadjusted medical expenditures by age group, and generalized estimating equations were used to estimate monthly median expenditures by age group with adjustment for comorbidity burden and functional status. Results: Of 34 317 patients aged 75 to 109 years (16 202 men [47.2%] and 18 115 women [52.8%]) who died between April 2014 and March 2018, 872 (2.5%) were aged 100 to 104 years (131 men [15.0%] and 741 women [85.0%]) and 78 (0.2%) were aged 105 to 109 years (fewer than 10 were men). The analysis of unadjusted medical expenditures in the last year of life showed a significant trend of lower expenditures for the older age groups; the median adjusted total expenditures during the 30 days before death by age group were $6784 (IQR, $4884-$9703) for ages 75 to 79 years, $5894 (IQR, $4292-$8536) for 80 to 84 years, $5069 (IQR, $3676-$7150) for 85 to 89 years, $4205 (IQR, $3085-$5914) for 90 to 94 years, $3522 (IQR, $2626-$4861) for 95 to 99 years, $2898 (IQR, $2241-$3835) for 100 to 104 years, and $2626 (IQR, $1938-$3527) for 105 to 109 years. The proportion of inpatients among all patients in the year before death also decreased with increasing age: 4311 of all 4551 patients aged 75 to 79 years (94.7%); 43 of all 78 patients aged 105 to 109 years (55.1%); 2831 of 2956 men aged 75 to 79 years (95.8%); 50.0% of men aged 105 to 109 years (the number is not reported owing to the small sample size); 1480 of 1595 women aged 75 to 79 years (92.8%); and 55.7% of women aged 105 to 109 years (the number of women is not reported to prevent back-calculation of the number of men). Specifically, 274 of 872 patients aged 100 to 104 years (31.4%) and 35 of 78 patients aged 105 to 109 years (44.9%) had not been admitted to a hospital in the year before death. Conclusions and Relevance: This cohort study found that medical expenditures in the last year of life tended to be lower for centenarians than for noncentenarians aged 75 years or older in Japan. The proportion of inpatients also decreased with increasing age. These findings may inform future health care services coverage and policies for centenarians.

A Gender Comparison of Bone Metabolic Changes After Gastric Cancer Surgery: A Prospective Observational Study.

BACKGROUND/AIM: This study was designed to investigate gender-related differences in changes in bone metabolism after gastric cancer surgery. PATIENTS AND METHODS: We prospectively recruited 47 patients (38 males and 9 females) who had early gastric cancer. The bone mineral density (BMD), serum levels of 1,25-dihydroxy vitamin D (1,25(OH)2VD), 25-hydroxy vitamin D (25(OH)VD), and estradiol (E2) were measured before and after surgery. RESULTS: BMD significantly decreased 12 months after surgery by median degrees of 3.4% and 3.9% in male and female patients, respectively (p<0.001 and p=0.023). There was no significant difference between both genders in the rate of change in BMD after surgery. The serum E2 level in male patients significantly increased by a median value of 22 pg/ml 12 months after gastrectomy (p=0.030). Both the serum 25(OH)VD and 1,25(OH)2VD levels remained nearly within the normal range throughout the observation period in both male and female patients. CONCLUSION: BMD significantly decreased within 12 months after gastrectomy in both male and female patients with gastric cancer, and there was no significant gender-related difference in the rate of change in BMD.

Long-term Outcomes of Therapeutic Endoscopic Retrograde Cholangiopancreatography for Choledocholithiasis in Patients ≥90 Years Old: A Multicenter Retrospective Study.

Objective The safety and prognosis of complete stone removal for the treatment of choledocholithiasis in older patients are unknown. This multicenter retrospective study assessed the outcomes of complete stone removal in elderly patients (≥90 years) with respect to the prognosis. Methods We divided patients who underwent endoscopic cholangiopancreatography for choledocholithiasis into two groups: complete stone removal or incomplete stone removal with plastic stent insertion. The patient characteristics, adverse events, number of endoscopic cholangiopancreatographies, overall survival rates, and disease-specific cumulative death were compared between the groups. Patients Two hundred and twenty-three participants ≥90 years old were included in the study, including 48 (22%) men and 175 (78%) women. The median age was 92 (range, 90-104) years old. There were 160 (72%) and 63 (28%) patients in the complete and incomplete groups, respectively. Results The age, performance status, comorbidities, severe complication rates, and stone diameter were comparable between the groups. The proportion of patients with at least 5 stones was significantly higher in the incomplete group than in the complete group [complete group: 8.1% (13/160) and incomplete group: 21% (13/63), p<0.01]. The overall survival rate was significantly higher in the complete group (p<0.01), while the disease-specific cumulative death rate was higher in the incomplete group (p<0.01). Conclusion Complete stone removal for choledocholithiasis may contribute to a better prognosis in elderly patients ≥90 years old.

Can Non-expert Physicians Use the Japan Narrow-band Imaging Expert Team Classification to Diagnose Colonic Polyps Effectively?

OBJECTIVES: In 2014, the Japan narrow-band imaging expert team (JNET) proposed the first unified colorectal narrow-band imaging magnifying classification system, the JNET classification. The clinical usefulness of this system has been well established in JNET member institutions, but its suitability for use by "non-expert physicians" (physicians with no expertise in the use of JNET classification) remains unclear. This study aimed to examine the clinical usefulness of the JNET classification by "non-expert physicians". METHODS: We retrospectively analyzed 852 consecutive patients who underwent screening colonoscopy following a positive fecal occult blood test between January 2017 and May 2018. Endoscopic results from colon polyp diagnosis by physicians who started using the JNET classification (JNET group) were compared with those of physicians who did not (control group). Mann-Whitney U test and Fisher's exact test were used to compare continuous and categorical variables, respectively. RESULTS: The median patient age was 68 years, and the male-to-female ratio was 1:0.84. When no lesions were found, the median withdrawal time was significantly different between groups (JNET group: 12 min; control group: 15 min; P < 0.01). The number of resected adenomas per colonoscopy was significantly higher in the JNET group (1.7) than in the control group (1.2; P < 0.01). Among the resected lesions, 8.9% in the JNET group and 17% in the control group were non-neoplastic lesions that did not require resection (P < 0.01). CONCLUSIONS: Colon polyp diagnosis using the JNET classification can reduce unnecessary resection during magnifying colonoscopy when conducted by "non-expert physicians".

Is complete stone removal for choledocholithiasis always necessary in extremely elderly patients?

BACKGROUND AND AIM: Endoscopic stone removal has some complications. Although the life expectancy of elderly patients has increased dramatically worldwide, little information is available on the necessity of complete endoscopic stone removal in extremely elderly patients. This study aimed to evaluate the safety and efficacy of complete endoscopic stone removal in extremely elderly patients. METHODS: All extremely elderly patients (>90 years) who underwent endoscopic stone removal for choledocholithiasis at our hospital between January 2012 and January 2017 were retrospectively evaluated. The included patients were divided into complete stone removal and incomplete stone removal groups. Complication rate, overall survival (OS), and disease-specific survival (DSS) rates were compared between the two groups. RESULTS: Overall, 73 patients were included in this study. The median number of stones was one (range, 0-10) and two (range, 1-12) (P = 0.043), while the median diameter of the largest stones was 9 (range, 0-27) and 14 (range, 5-46) mm (P = 0.001) in the complete and incomplete stone removal groups, respectively. During the follow-up period, OS was 60% and 39% and DSS was 95% and 97% in the complete and incomplete stone removal groups, respectively. Kaplan-Meier analysis found no significant difference in OS and DSS between the two groups (P = 0.052 and P = 0.646, respectively). CONCLUSION: Complete stone removal might not always be necessary in extremely elderly patients aged ≥90 years.

Compositional analysis of the glycosaminoglycan family in velvet antlers of Sika deer (Cervus nippon) at different growing stages.

Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.

Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111.

We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)ßGalNAc6S(1-4)ßGlcA(1-]2 and CS-DD [-3)ßGalNAc6S(1-4)ßGlcA2S(1-]2 which possess sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. We also analyzed interactions among CS-CC and CS-DD and the antibodies 2H6 and LY111, both of which are known to bind with CS-A, while CS-DD was shown for the first time to bind with both antibodies.

Synthesis of a biotinylated keratan sulfate tetrasaccharide composed of dimeric Galß1-4GlcNAc6Sß.

We successfully synthesized the biotinylated keratan sulfate tetrasaccharide, Galß1-4GlcNAc6Sß1-3Galß1-4GlcNAc6Sß in a stereocontrolled manner. The suitably protected Galß1-4GlcNPhth unit was converted to the corresponding donor and acceptor. Optimization in 2 + 2 coupling using AgOTf, CuBr2, and n-Bu4NBr in CH3NO2 at a low temperature afforded the desired tetrasaccharide that suppressed glycal formation. The subsequent chemoselective removal of the protecting group at O-6 of two GlcNAcs, sulfation, and deprotection procedures as well as biotinylation gave the target compound.

Total synthesis of TMG-chitotriomycin based on an automated electrochemical assembly of a disaccharide building block.

The total synthesis of TMG-chitotriomycin using an automated electrochemical synthesizer for the assembly of carbohydrate building blocks is demonstrated. We have successfully prepared a precursor of TMG-chitotriomycin, which is a structurally-pure tetrasaccharide with typical protecting groups, through the methodology of automated electrochemical solution-phase synthesis developed by us. The synthesis of structurally well-defined TMG-chitotriomycin has been accomplished in 10-steps from a disaccharide building block.

Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

Early Gastric Cancer Just above a Heterotopic Pancreas.

We report the first case of early gastric cancer just above a heterotopic pancreas for which the differential diagnosis was carcinoma arising from heterotopic pancreas. Routine upper gastrointestinal endoscopy in an 83-year-old man with sigmoid colon cancer revealed a gastric cancer in the lesser curvature of the antrum. Endoscopic ultrasonography (EUS) for evaluating the depth of tumor invasion revealed a hypoechoic mass in the submucosal layer. The depth of tumor invasion was diagnosed as muscularis propria. Distal gastrectomy and sigmoidectomy were performed. Histologically, the resected specimen of the stomach unexpectedly revealed a heterotopic pancreas just below the gastric cancer. They were not linked, and the heterotopic pancreas had no dysplasia. The gastric cancer had slightly invaded the submucosa. The hypoechoic mass on EUS was not the invasive tumor but the heterotopic pancreas. The preoperative staging of the gastric cancer on EUS was confounded by the presence of the heterotopic pancreas just below the gastric cancer.

Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme.

Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4-8). This occurs either directly within certain tissues or by interconversion to menadione (K(3)), followed by prenylation to MK-4 (refs 9-12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K(3) into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d(7)) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.