Effect of 4 hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: a comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of 4 hFAs-10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA)-on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9,12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity level. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Disseminated histiocytic sarcoma in a squirrel monkey (Saimiri sciureus).

A 14-years-old squirrel monkey was euthanized due to weakness. Histopathological examination revealed multifocal growth of oval cells with severe atypia in the liver, spleen, and bone marrow. The neoplastic cells were positive for histiocytic markers (Iba1, HLA-DR, CD204). This is the fourth case of histiocytic sarcoma in non-human primates.

Characterization of calcium ion sensitive region for ß-mannanase from Streptomyces thermolilacinus.

Despite the widespread industrial applications of ß-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on ß-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding ß-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.

Predictive Factors for Successful Treatment of Deep Incisional Surgical Site Infections following Instrumented Spinal Surgeries: Retrospective Review of 1832 Cases.

BACKGROUND: Surgical site infection (SSI) is a major complication in spinal instrumentation that is often difficult to treat. The purpose of this study was to identify and determine prognostic indicators for successful treatment of spine instrumentation SSI. METHODS: Retrospectively, spine surgery cases were examined on SSI diagnosis. Post-instrumentation SSI patients were categorized as "Successful" if SSI subsided after single debridement. Patients in whom SSI did not subsided and/or required removal of instrumentation were classified as "Challenging". We investigated the relation of treatment outcomes to patients and treatment factors. RESULTS: A total of 1832 spinal instrumentation cases were recognized with 44 (2.40%) SSI cases. White blood cell count, C-reactive protein (CRP) levels, causative bacteria (i.e., S. Aureus or MRSA), trauma injury, and early-stage antimicrobial agent sensitivity correlated with treatment prognosis. Multivariate analysis highlighted CRP levels and applying early-stage sensitive antibiotics as potential impactful predictive factors for successful treatment. CONCLUSIONS: Our results demonstrated that early selection of sensitive antimicrobial agents is critical and emphasizes the potential for early-stage classification methods such as Gram staining. Additionally, S. Aureus and MRSA SSI formed significantly more challenging infections to treat, thus requiring consideration when deciding on instrumentation retention. These factors offer promising aspects for further large-scale studies.

MS/MS fragmentation-guided search of TMG-chitooligomycins and their structure-activity relationship in specific ß-N-acetylglucosaminidase inhibition.

The reducing tetrasaccharide TMG-chitotriomycin (1) is an inhibitor of ß-N-acetylglucosaminidase (GlcNAcase), produced by the actinomycete Streptomyces anulatus NBRC13369. The inhibitor shows a unique inhibitory spectrum, that is, selectivity toward enzymes from chitin-containing organisms such as insects and fungi. Nevertheless, its structure-selectivity relationship remains to be clarified. In this study, we conducted a structure-guided search of analogues of 1 in order to obtain diverse N,N,N-trimethylglucosaminium (TMG)-containing chitooligosaccharides. In this approach, the specific fragmentation profile of 1 on ESI-MS/MS analysis was used for the selective detection of desired compounds. As a result, two new analogues, named TMG-chitomonomycin (3) and TMG-chitobiomycin (2), were obtained from a culture filtrate of 1-producing Streptomyces. Their enzyme-inhibiting activity revealed that the potency and selectivity depended on the degree of polymerization of the reducing end GlcNAc units. Furthermore, a computational modeling study inspired the inhibitory mechanism of TMG-related compounds as a mimic of the substrate in the Michaelis complex of the GH20 enzyme. This study is an example of the successful application of a MS/MS experiment for structure-guided isolation of natural products.

Peptide bond formation by aminolysin-A catalysis: a simple approach to enzymatic synthesis of diverse short oligopeptides and biologically active puromycins.

A new S9 family aminopeptidase derived from the actinobacterial thermophile Acidothermus cellulolyticus was cloned and engineered into a transaminopeptidase by site-directed mutagenesis of catalytic Ser(491) into Cys. The engineered biocatalyst, designated aminolysin-A, can catalyze the formation of peptide bonds to give linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including the l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis and not a reverse reaction of hydrolysis. The aminolysin system will be a beneficial tool for the preparation of structurally diverse peptide mimetics by a simple approach.

Facile Preparation of Purified Sinapate Ethyl Ester from Rapeseed Meal Extracts Using Cation-exchange Resin in Dual Role as Adsorber and Catalyst.

In this study, cation-exchange resin was used to prepare an esterified antioxidant, sinapate ethyl ester (SE), using ethanolic extracts from rapeseed. A concentration of sinapic acid using the cation-exchange resin in 80% ethanol (aq) and subsequent interesterification of the extract in ethanol using the same resin afforded a product with a purity of 64 wt% and 100% of SE yield. Moreover, after purification using preparative thin-layer chromatography, almost 100 wt% purity was obtained. In an auto-oxidation test, purified SE conferred a much higher antioxidative effect on the bulk oil, emphasising the effectiveness of the protocol using cation-exchange resin for the purification.

The Enzymatic Preparation of Human Milk Fat Substitute Intermediate Rich in Palmitic Acid at sn-2 Position and Low-Unsaturated Fatty Acids at sn-1(3) Positions from Palm Oil Substrate.

The lipid products that consist of structured lipids rich in palmitic acid (16:0) at the sn-2 position of triacylglycerol (TAG) and rich in low-unsaturated fatty acids (FAs) (LUFAs), such as oleic acid; 18:1 and linoleic acid; 18:2 at the sn-1(3) positions, are useful intermediates for manufacturing human milk fat substitute (HMFS), which contains functional lipid components. In this study, the HMFS intermediate (HMFS-IM) was enzymatically prepared from palm oil without using other oil sources. First, the amount of 16:0 at the sn-2 position of TAG substrate was enhanced from 18.9% to more 34.5% via a random esterification reaction using a non-stereospecific lipase, Novozym® 435, to produce a random-palm substrate. Consequently, 2-monoacylglycerol (2-MAG) rich in 16:0 at the sn-2 position over 88%, together with the FA ethyl ester substrates rich in LUFAs, such as 18:1-Et and 18:2-Et above 93.5% was prepared through ethanolysis reaction using the same lipase from the random-palm substrate and by purification with urea complexation, respectively. As the preferred modified method, a continuous use of the same lipase to these reactions were achieved while reducing the usage of enzyme to half. Finally, an HMFS-IM rich in 16:0 at the sn-2 position more than 60% and LUFA at sn-1(3) positions was prepared using these palm oil-based products, including random-palm, palm-Et, and 2-MAG, via the interesterification reaction using a 1,3-stereospecific lipase, Lipozyme® RM-IM. Thus, HMFS-IM was successfully prepared by palm oil materials with a 65 wt% usage ratio. The concept described in this study will be useful for HMFS manufacturing from a single natural oil substrate, which is not initially rich in 16:0 at the sn-2 position.

Antiadipogenic Effects of Different Molecular Forms of Conjugated Linoleic Acid on 3T3-L1 Cells: Comparison between Free Fatty Acid and Phosphatidylcholine Forms.

The antiadipogenic activity of conjugated linoleic acids (CLA) in the form of phosphatidylcholine-bound (CLA-PC) or free fatty acids (FFA; CLA-FFA) was evaluated using 3T3-L1 adipocytes. Phosphatidylcholine from soya (soy-PC) was used as the comparison of PC form. Both the lipid accumulation and activity of glycerol-3-phosphate dehydrogenase were measured to determine lipogenesis, whereas the glycerol content was measured to evaluate lipolysis. The CLA uptake also measured to find out the utilization of CLA by the cells. As a results, lipid accumulation in 3T3-L1 adipocytes was inhibited in a dose-dependent manner following treatment with CLA-PC (50-400 µM). Both CLA-PC and soy-PC significantly suppressed lipid accumulation compared with CLA-FFA, even though the amount of CLA in CLA-PC was a half than CLA-FFA. The CLA uptake of PC form was superior to FFA form, however, no difference was noted between CLA-PC and soy-PC. These forms exerted their antiadipogenic activity via the suppression of lipogenesis, and not by increasing lipolysis. Short-term treatment, especially in the middle stage of differentiation, was more effective than long-term treatment; especially for CLA-FFA. The antiadipogenic effect of CLA-PC was partially attributed to the chemical structure of the PC molecule. These results provide important information for the utilization of physiologically functional fatty acids and particularly CLA in the food and medical fields.

Prolyl aminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides by its S144C variant.

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.

Upper cervical spine injuries: age-specific clinical features.

BACKGROUND: There are few reports on the age-specific clinical features of upper cervical spine injury. To identify these age-specific changes, we reviewed 103 patients with upper cervical spine injury. METHODS: We subdivided the patients into four groups according to age: 12 patients were classified as old elderly (> or = 75 years), 18 patients as young elderly (65-74 years), 67 patients as young adults (18-64 years), and 6 patients as adolescents (< or = 17 years). Data were collected on injury etiology, mortality, neurological deficit, distribution and pattern of injury, degenerative changes of each joint in the upper cervical spine and disc in the cervical spine, and osteoporosis of the axis. RESULTS: The proportion of patients with a simple fall as the etiology of their injury showed a statistically significant trend to increase with aging. Although no specific distribution or pattern of injury was statistically elucidated, we were able to detect some common features. A high proportion of the old elderly group (5/12) had a type II odontoid fracture. Body fracture of the axis tends to be frequent in the young elderly and traumatic spondylolisthesis and hyperextension teardrop fracture to be frequent in the young adults. In adolescents, a traffic accident as an unbelted rear seat passenger was the most frequent etiology. Among the 103 patients, 16 died during the initial hospitalization. The mortality rate for upper cervical spine injury was similar in all groups. CONCLUSIONS: In the old elderly, stiffness of the lower cervical spine, caused by degenerative changes and osteopenia, might contribute to upper cervical spine injury in response to low-energy trauma. The disproportion of degenerative change in joints of the upper cervical spine might also contribute to the high frequency of type II odontoid fracture. In young adults, high-energy and hyperextension injury was the most frequent cause of upper cervical spine injury.

Highly efficient preparation of 1-lysophosphatidylcholine via high proportion of Novozym® 435 (lipase B from Candida antarctica)-catalyzed ethanolysis.

Efficient preparation methods for 1-lysophosphatidylcholine (1-LPC), a physiologically important compound, are lacking. Here, we established a method for 1-LPC preparation via Novozym® 435 (a lipase B from Candida antarctica)-catalyzed ethanolysis. Novozym® 435 showed sn-1 regiospecificity to phosphatidylcholine, although it does not exhibit regiospecificity to triacylglycerol. In particular, quantitative 1-LPC yields (96.5 ±â€¯0.2 mol%) were reliably obtained in the presence of Novozym® 435 (100 wt% of PC), 97 % ethanol, in 72 h at 40 °C. During the reaction, acyl migration from 1-LPC to 2-LPC was rare. This novel synthetic method is expected to expand the practical applications of 1-LPC.

Enzymatic Preparation and Oxidative Stability of Human Milk Fat Substitute Containing Polyunsaturated Fatty Acid Located at sn-2 Position.

The development of human milk fat substitutes (HMFSs), rich in palmitic acid (16:0) at the sn-2 position of triacylglycerol (TAG) and rich in unsaturated fatty acids (FAs) (oleic acid, 18:1 and linoleic acid, 18:2) at the sn-1(3) positions, has gained popularity. In this study, HMFSs containing polyunsaturated fatty acids (PUFAs) predominantly at the sn-2 position were prepared, and their oxidation stabilities were compared. First, a non-PUFA-containing HMFS (NP-HMFS) was produced by enzymatic reactions using Novozyme® 435 and Lipozyme® RM-IM as the enzymes and lard as the raw material. Second, HMFSs, containing 10 % PUFA at the sn-2 or sn-1(3) position, were individually prepared by enzymatic reactions using lard and fish oil as raw materials. Here, sn-2-PUFA-monoacylglycerol (MAG) was extracted from the reaction solution using a mixture of hexane and ethanol/water (70:30, v/v) to produce high-purity sn-2-PUFA-MAG with 78.1 % yield. For the PUFA-containing HMFS substrates, comparable oxidation stability was confirmed by an auto-oxidation test. Finally, HMFSs containing 10 % or 2 % sn-1,3-18:1-sn-2-PUFA-TAG species were prepared by enzymatic reactions and subsequent physical blending. The oxidative stability of sn-1,3-18:1-sn-2-PUFA-HMFS was two-fold higher than that of 1/2/3-PUFA-HMFS in which each PUFA was located without stereospecific limitations in TAG. The removal of PUFA-TAG molecular species with higher concentrations of unsaturated units had a significant effect. In addition, the oxidation stability increased with the addition of tocopherol as an antioxidant. Thus, the combined use of two strategies, that is, the removal of PUFA-TAG molecular species with high concentrations of unsaturated units and the addition of antioxidants, would provide a PUFA-containing HMFS substrate with high oxidative stability.

Effect of Molecular Form of Conjugated Linoleic Acid on Oxidative Stability : Comparison of Triacylglycerol and Phosphatidylcholine Form.

The health benefits of conjugated linoleic acid (CLA), a functional lipid with anti-cancer, anti-obesity, and hypotensive activity, have garnered increasing attention. The current study was conducted to determine the oxidative stability of CLA in the form of triacylglycerol (CLA-TAG) and phosphatidylcholine (CLA-PC) at the sn-2 position. Oxidation was performed at 30°C or 40°C in the dark. Hydroperoxides, as the primary oxidation products, were analyzed using diphenyl-1-pyrenylphosphine. Thiobarbituric acid reactive substances (TBARS) and volatile compounds were monitored as secondary oxidation products. The results suggest that CLA-PC was more stable against oxidation than CLA-TAG from the perspective of suppression of the generation of hydroperoxides and TBARS. However, CLA-PC produced more volatile compounds than CLA-TAG. We suggest that choline was released during the oxidation of CLA-PC, and acted as an antioxidant. The ensuing reaction between choline and hydroperoxide induced the generation of volatile compounds such as pentanal, hexanal, and heptanal.

Effect of alpha-tocopherol on the synthesis of phosphatidylglycerol catalyzed by phospholipase D in an aqueous system.

Phosphatidylglycerol (PG) was synthesized from several phosphatidylcholines (PCs) via phospholipase D (PLD)-catalyzed transphosphatidylation in an aqueous system. The yield of PG were 71 and 68 mol% from soybean PC and egg yolk PC, respectively, under the optimum reaction conditions of 50 micromol PC, 10 mmol glycerol, 3 ml of acetate buffer, 1.6 U PLD, and 30 micromol CaCl(2) at 37 degrees C for 48 h. In case of salmon roe PC with 14.3% eicosapentaenoic acid and 26.8% docosahexaenoic acid, the PG yield increased to 94 mol% by addition of 46 mumol alpha-tocopherol, although the PG yield was only 10% in absence of alpha-tocopherol.

Upper cervical spinal cord tumors: review of 13 cases.

BACKGROUND: Clinical features of upper cervical spinal cord tumors are not clear because there have been too few published reports. The purpose of this study was to review the clinical features of these tumors. METHODS: We reviewed 13 patients who underwent surgery for an upper cervical spinal cord tumor. Data regarding age, sex, duration and type of symptoms, levels, topographical locations, surgical results, and histological features were investigated retrospectively. RESULTS: Of the 13 tumors, 5 were at C1/2, 7 at C2/3 and 1 at C1-3. Topographically, 9 of the 13 tumors were dumbbell shaped, and all 5 at C1/2 had this shape. The initial symptom was occipital pain in three patients, numbness or pain of the extremity in nine, and clumsiness of the upper extremity in one. The average duration from initial symptom to surgery was 14.9 months. There were no major surgical complications, but there were three cases of postoperative cervical kyphosis. These three patients underwent tumorectomy with total laminectomy of C2. Tumor recurrence was seen in three patients for reasons thought to be the same as tumors at other levels, being residual meningiomas lying ventral to the cord and extraforaminal neurinomas. CONCLUSIONS: Spinal cord tumors in the upper cervical region tend to progress as dumbbell tumors. As all the neurinomas had this shape (Eden type 2 or 3) at C1/2, this anatomy might favor progression to the extradural and extraforaminal spaces.However, it also allows total removal of the tumor via a posterior approach. To maintain postoperative cervical alignment, the surgeon should select the least invasive approach to the paraspinal muscles attached to the spinous process of C2.

A Patient with Pediatric Septic Arthritis of the Hip Complicated with Osteomyelitis Responds to Clindamycin.

A 5-year-old girl was referred to our institution with complaints of right hip pain and fever (39.0°C); blood tests and magnetic resonance imaging (MRI) were performed. Blood biochemical results revealed a high inflammatory reaction. MRI results revealed inflammation in the hip joint. She was diagnosed with pediatric septic arthritis of the hip, which required arthrotomy and lavage drainage. Treatment with intravenous vancomycin was initiated on the assumption that the causative bacterial species was Staphylococcus aureus. However, Group A Streptococcus pyogenes (GAS) was subsequently isolated from an intraoperative sample, and the antimicrobial agent was switched to aminobenzylpenicillin (ABPC). Clindamycin (CLDM) was added to the treatment regimen 14 days after surgery as MRI indicated the development and spread of osteomyelitis. Three months post-surgery, MRI findings indicated that the osteomyelitis had resolved and antimicrobial therapy was discontinued. To prevent the spread of osteomyelitis, a combination of CLDM and ABPC should be considered at an early stage, particularly in pediatric patients with GAS-induced septic arthritis.

Significance of leptin and high-molecular weight adiponectin in the general population of Japanese male adolescents.

Adipokines play crucial roles in obesity-related insulin resistance in adults, but little is known in the general adolescent population. This study was designed to investigate the relationships between adipokines and metabolic parameters, the insulin resistance index, and proinflammatory cytokines in the general population of Japanese male adolescents. We studied 662 Japanese male high school students aged 16 to 17 years and 282 healthy Japanese male adults aged 30 to 61 years who received annual health checkups. High-molecular weight (HMW) adiponectin levels were significantly lower in adolescents (4.18 +/- 2.24 microg/mL) than in adults (4.84 +/- 3.20 microg/mL), despite body mass index (BMI) being significantly lower in adolescents. The HMW adiponectin levels correlated negatively with BMI and the homeostasis model assessment of insulin resistance index (HOMA-IR) in adults. In adolescents, HMW adiponectin correlated negatively with BMI and waist circumference, but not with HOMA-IR or other metabolic parameters except high-density lipoprotein cholesterol. Leptin levels correlated positively with HOMA-IR, triglycerides, tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1 and negatively with high-density lipoprotein cholesterol even after adjustment for BMI. These findings suggest that serum leptin is a more useful biomarker of fat accumulation-related insulin resistance, inflammation, and metabolic abnormalities than HMW adiponectin in the general population of male adolescents. The inverse correlation between adiponectin and insulin resistance may manifest in the later phase of obesity development.

Synthesis of phosphatidylated-monoterpene alcohols catalyzed by phospholipase D and their antiproliferative effects on human cancer cells.

In order to prepare functional phospholipids in the medical and pharmaceutical fields, perillyl alcohol, myrtenol, and nerol were transphosphatidylated via phospholipase D in an aqueous system. The yields of phosphatidyl-perillyl alcohol, -myrtenol, and -nerol were 79 mol %, 87 mol %, and 91 mol %, respectively. The synthetic phosphatidylated monoterpenes showed a markedly antiproliferative effect on human prostate PC-3 and human leukemia HL-60 cells at 100 microM, while the free monoterpene alcohols had no effect at 400 microM.

Combination of C-reactive protein and high molecular weight (HMW)-adiponectin reflects further metabolic abnormalities compared with each of them alone in Japanese type 2 diabetic subjects.

OBJECTIVE: Serum C-reactive protein (CRP) and adiponectin levels predict atherosclerosis and coronary heart disease. However, the efficacy of the combination of both markers remains unknown. In the present study, we investigated whether the combination of CRP and adiponectin is associated with further metabolic abnormalities compared to each of them alone. RESEARCH DESIGN AND METHODS: Eighty-three Japanese type 2 diabetic outpatients participated in this study. We measured serum high-sensitive CRP and high molecular weight (HMW)-adiponectin, and investigated their relationship with various metabolic parameters. RESULTS: In univariate analysis, CRP was significantly correlated with diastolic blood pressure and HDL-cholesterol. On the other hand, HMW-adiponectin was significantly correlated with systolic (SBP) and diastolic blood pressure, plasma glucose, HDL-cholesterol, triglycerides and HOMA-IR, but not with CRP. We then classified the subjects into three groups: low CRP and high HMW-adiponectin levels (low risk group, 19%), high CRP and low HMW-adiponectin levels (high risk group, 22%), and others. In Spearman rank correlation coefficient analysis, this classification was significantly associated with a larger number of metabolic risk factors: SBP, glucose, HbA1c, LDL-cholesterol, HDL-cholesterol, triglycerides and HOMA-IR, compared with classification by CRP or HMW-adiponectin alone. CONCLUSION: These results suggest that combination of CRP and HMW-adiponectin reflects further metabolic abnormalities compared with each of them in type 2 diabetic subjects. The combined measurement of both markers may be useful to detect cardiovascular high risk patients.