Pelvic incidence is a risk factor for lower instrumented vertebra failure in adult spinal deformity patients who underwent corrective fusion terminating at the L5 vertebra.

BACKGROUND: Factors related to postoperative mechanical failure after long fusion with lower instrumented vertebra (LIV) at L5 have not been well investigated. Elucidating such factors may allow us to perform alternatives to spinopelvic fusion for adult spinal deformity (ASD) cases. We investigated the incidence and risk factors of LIV failure in patients with ASD who underwent surgical treatment of long corrective fusion until the L5 vertebrae. METHODS: Between 2009 and 2018, 52 patients who underwent corrective fusions to L5 were followed-up for at least one-year. We evaluated the associated patient factors for LIV failure which include loosening of the pedicle screw of LIV, fracture of LIV, distal junctional kyphosis (DJK). RESULTS: The mean age of the participants was 71.2 ± 7.59 (range, 44-84). LIV failure occurred in 20 patients (38.5%), and 6 patients (11.5%) underwent secondary surgery for caudal segments. The mean pelvic incidence (PI) was 52.5 ± 9.8 in the failure group versus 45.3 ± 11.4 in non-failure group (P = 0.02) and pelvic tilt (PT) was 39.1 ± 9.0 versus 32.4 ± 13.0. There were no significant differences in sex, age, body mass index, number of levels fused, and other radiographic data. Logistic regression analysis that included T1 pelvic angle, PT, PI - postoperative LL and PI also identified PI as the only significant determinant of LIV failure (OR = 1.07, P = 0.034). Receiver operating characteristic analysis demonstrated that a PI over 50.0° was associated with LIV failure (sensitivity 63%, specificity 70%, AUC 0.694). CONCLUSION: LIV failure was frequently observed after long corrective fusion for patients with ASD. High PI was found to be a significant risk factor for the LIV failure.

Munc13b stimulus-dependently accumulates on granuphilin-mediated, docked granules prior to fusion.

The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner.Key words: docking, insulin, live cell imaging, priming, TIRF microscopy.

Prognostic factors for neurological outcome after anterior decompression and fusion for proximal-type cervical spondylotic amyotrophy - A retrospective analysis of 77 cases.

BACKGROUND: Decompression through an anterior approach is theoretically effective for the surgical treatment of cervical spondylotic amyotrophy (CSA), because the pathology usually locates at the anterior side. However, most previous studies investigated posterior surgery or a mix of anterior surgery and posterior surgery in their investigation. Only a few small case series have investigated the surgical outcomes of anterior decompression and fusion (ADF). Therefore, we conducted a multicenter retrospective study that included patients who underwent ADF for proximal-type CSA. METHODS: We analyzed the outcomes of 77 consecutive spinal surgeries performed on proximal-type CSA patients who underwent ADF. Preoperative and postoperative manual muscle tests (MMT) and the patients' backgrounds, radiological findings, and complications were reviewed. We divided the cases into two groups, good-outcome group (MMT improvement â‰§ 2 or improved to MMT 5) and poor-outcome group (others) and evaluated the prognostic factors for outcomes. RESULTS: Of the 77 patients, 48 (62%) showed good neurological outcome. Multiple compressive lesions at anterior horn (AH) and/or ventral nerve roots (VNRs) were detected in 66 patients (85.7%) on the magnetic resonance images. The patients with a single compressive lesion at VNR or AH tended to show good neurological recovery when compare to those with multiple lesions. Age and duration of symptoms were related to the poor outcome in univariate analysis. Duration of symptoms was an independent factor associated with postoperative neurological outcome. The cut-off value for poor outcome was 7.0 months for the symptom duration (sensitivity: 79%, specificity: 54%, area under the curve: 0.69). CONCLUSIONS: Patients with proximal-CSA were more likely to have multiple compressive lesions at an AH and/or a VNR. The prognostic factor for poor neurological outcome was duration of symptoms of ≥7 months.

Gene expression analysis of enzymes of the carotenoid biosynthesis pathway involved in ß-cryptoxanthin accumulation in wild raspberry, Rubus palmatus.

ß-cryptoxanthin (ß-Cry), a xanthophyll, is unlike other abundant carotenoids, such as α-carotene, ß-carotene, lycopene, lutein, and zeaxanthin. It is not found in most fruits or vegetables but is found only in specific fruits, such as hot chili pepper, persimmon, and citrus fruits. Because recent reports suggest that ß-Cry intake is beneficial to human health, the xanthophyll requires further investigation. Although ß-Cry accumulates in the fruit of wild raspberry, Rubus palmatus, it is not present in cultivated raspberry. In the present study, two wild raspberry species were studied-R. palmatus, which accumulates ß-Cry in the fruit, and R. crataegifolius, which does not accumulate ß-Cry. Four carotenoid biosynthetic enzymes derived from these two species were analyzed-phytoene synthase (PSY), lycopene ß-cyclase (LCYb), ß-carotene hydroxylase (HYb), and zeaxanthin epoxidase (ZEP). Expression levels of their genes were also assessed to elucidate mechanism underlying ß-Cry accumulation. Partial gene sequences of RubPSY, RubLCYb, RubHYb, and RubZEP, isolated from immature raspberry fruits of R. palmatus, were used as probes for Northern blot analysis. RubZEP expression ceased as the fruits matured, possibly because of reduced production of zeaxanthin. ß-Cry is considered to be an intermediate compound that accumulates in the mature fruits of R. palmatus. High expression of RubPSY was detectable in the mature fruits of R. crataegifolius, and the expression of RubLCYb, RubHYb, and RubZEP was detectable during all stages of fruit maturation. In contrast, ß-Cry was absent in the mature fruits of R. crataegifolius.

Effects of Valsalva Maneuver by Different Breath-holding Techniques to Detect Pulmonary Thromboembolism in a Contrast-enhanced Computed Tomography Examination.

The purpose of this study is to measure the hemodynamics on the effect of Valsalva maneuver aiming at pulmonary thromboembolism (PTE) using 2-dimensional (2D) phase contrast imaging of magnetic resonance image (MRI), Philips Ingenia 3.0-tesla (T). The maximal inspiration reduced the blood flow rate in various degrees at all measurement positions, superior vena cava (SVC), inferior vena cava (IVC), pulmonary artery (PA), ascending aorta (AA), and descending aorta (DA). This result suggests that the contrast effect in the PA might become weak during general PA phase to give a substantial influence of Valsalva maneuver in the condition after maximum inspiration. A contrast-enhanced computed tomography (CT) examination aiming at detection for PTE should be scanned without an advance maximum inspiration.

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B' methyltransferase family in Coffea arabica.

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-(14)C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B'-MTs. It was concluded that CTgSs have strict substrate specificity. The K(m) values of CTgS1 and CTgS2 were 121 and 184µM with nicotinic acid as a substrate, and 68 and 120µM with S-adenosyl-L-methionine as a substrate, respectively.

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis.

The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.

Purification and properties of a nuclease from the fruit body of Tricholoma matsutake.

A nuclease was purified from the fruit body of Tricholoma matsutake. The molecular mass was 38 kDa. The optimum pH of the nuclease was about 8.0. Its activity was inhibited by GTP. The nuclease cleaved RNA and heat-denatured DNA endonucleolytically to produce 5'-mononucleotide, and showed 3'-nucleotidase activity. The amino acid sequence up to seven amino acids from the N-terminus was NH(2)-APPPSSN.

Essential region for 3-N methylation in N-methyltransferases involved in caffeine biosynthesis.

The caffeine biosynthetic pathway is composed of three methylation steps, and N-methyltransferase catalyzing each step has high substrate specificity. Since the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), theobromine synthase, and caffeine synthase are highly homologous to each other, these substrate specificities seem to be determined in a very restricted region. The analysis of site-directed mutants for CmXRS1 that naturally acts at the initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 3-N methylation needs a histidine residue at corresponding position 161 in the CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze the first and second methylation steps in caffeine biosynthesis.

Expression for caffeine biosynthesis and related enzymes in Camellia sinensis.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid that is present in high concentrations in the tea plant Camellia sinensis. Caffeine synthase (CS, EC 2.1.1.160) catalyzes the S-adenosyl-L-methionine-dependent N-3- and N-1-methylation of the purine base to form caffeine, the last step in the purine alkaloid biosynthetic pathway. We studied the expression profile of the tea caffeine synthase (TCS) gene in developing leaves and flowers by means of northern blot analysis, and compared it with those of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), and S-adenosyl-L-methionine synthase (SAMS, EC 2.5.1.6). The amount of TCS transcripts was highest in young leaves and declined markedly during leaf development, whereas it remained constant throughout the development of the flower. Environmental stresses other than heavy metal stress and plant hormone treatments had no effect on the expression of TCS genes, unlike the other three genes. Drought stress suppressed TCS gene expression in leaves, and the expression pattern mirrored that of the dehydrin gene. The amounts of TCS transcripts increased slightly on supply of a nitrogen source. We discuss the regulation of TCS gene expression.

Melanophilin Accelerates Insulin Granule Fusion without Predocking to the Plasma Membrane.

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic ß-cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric guanosine-5'-triphosphatase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic ß-cells both exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca2+]i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages.

Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-ß receptor, increases lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c- adipose tissue macrophages (ATMs) into GDF3-producing CD11c+ macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.

Rabring7, a novel Rab7 target protein with a RING finger motif.

Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis.

Xanthine Alkaloids: Occurrence, Biosynthesis, and Function in Plants.

Caffeine is a xanthine alkaloid found in non-alcoholic beverages such as tea, coffee, and cocoa. It was discovered in tea and coffee in the 1820s, but it was not until 2000 that details of molecular events associated with caffeine biosynthesis began to be unraveled. Reviewed are the occurrence of xanthine alkaloids in the plant kingdom and the elucidation of the caffeine biosynthesis pathway, providing details of the N-methyltransferases, belonging to the motif B' methyltransferase family, which catalyze three steps in the four-step pathway leading from xanthosine to caffeine. Pathways for the metabolism and degradation of xanthine alkaloids are discussed, although as yet the genes and enzymes involved have not been isolated. This chapter also considers the in planta role of caffeine in chemical defense that has been demonstrated using transgenic caffeine-forming tobacco and chrysanthemum plants, which are resistant to attack by pathogens and herbivores. Finally, future research is considered that might lead to the production of naturally decaffeinated beverages and agricultural crops that contain elevated levels of "natural" pesticides.

Protodioscin, Isolated from the Rhizome of Dioscorea tokoro Collected in Northern Japan is the Major Antiproliferative Compound to HL-60 Leukemic Cells.

BACKGROUND: The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. METHODS: The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. RESULTS: PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. CONCLUSION: We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.

Genome wide cDNA-AFLP analysis of genes rapidly induced by combined sucrose and ABA treatment in rice cultured cells.

We identified 27 genes induced by combined sucrose and ABA treatment from rice cultured cells with cDNA-AFLP. Thirteen of these up-regulated genes were induced 30 min after the co-treatment. This suite of genes includes starch biosynthesis related genes. Type A genes were expressed only in the presence of both sucrose and ABA. Type B genes were expressed in the presence of sucrose or ABA and the expression was dramatically enhanced by the co-treatment of sucrose and ABA. These results indicate that multiple steps of starch biosynthesis and other processes may be regulated by at least two different pathways.

Granuphilin exclusively mediates functional granule docking to the plasma membrane.

In regulated exocytosis, it is generally assumed that vesicles must stably "dock" at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca(2+)-triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic ß cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules.

Rabring7: a target protein for rab7 small g protein.

Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to coordinate the individual step of vesicle traffic. Rabring7 (Rab7-interacting ring finger protein) is a Rab7 target protein that has been isolated using a CytoTrap system. This protein shows no homology with RILP, which has been reported as another Rab7 target protein. Rabring7 is recruited efficiently to late endosome/lysosome by the GTP-bound form of Rab7. Exogenous expression of Rabring7 not only affects epidermal growth factor degradation but also induces the perinuclear aggregation of lysosomes and the increased acidity in the lysosomes. This chapter describes the procedures for the isolation of Rabring7 with a CytoTrap system, the analysis of the Rab7-Rabring7 interactions, and the properties of Rabring7.

Biosynthesis of caffeine underlying the diversity of motif B' methyltransferase.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are well-known purine alkaloids in Camellia, Coffea, Cola, Paullinia, Ilex, and Theobroma spp. The caffeine biosynthetic pathway depends on the substrate specificity of N-methyltransferases, which are members of the motif B' methyl-transferase family. The caffeine biosynthetic pathways in purine alkaloid-containing plants might have evolved in parallel with one another, consistent with different catalytic properties of the enzymes involved in these pathways.

The first committed step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closely homologous to caffeine synthases in coffee (Coffea arabica L.).

In coffee and tea plants, caffeine is synthesized from xanthosine via a pathway that has three methylation steps. We identified and characterized the gene encoding the enzyme for the first methylation step of caffeine biosynthesis. The full-length cDNA of coffee tentative caffeine synthase 1, CtCS1, previously isolated by the rapid amplification of cDNA ends was translated with an Escherichia coli expression system and the resultant recombinant protein was purified using Ni-NTA column. The protein renamed CmXRS1 has 7-methylxanthine synthase (xanthosine:S-adenosyl-L-methionine methyltransferase) activity. CmXRS1 was specific for xanthosine and xanthosine 5'-monophosphate (XMP) could not be used as a substrate. The K(m) value for xanthosine was 73.7 microM. CmXRS1 is homologous to coffee genes encoding enzymes for the second and third methylation steps of caffeine biosynthesis.