Molecular characterization of GPR84 in domestic cats.

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83-89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein-protein interaction with FFAR2/GPR43 on the cell membrane.

[A Case of Metastatic Prostate Cancer with Neuroendocrine Differentiation with Long-Term Survival after Multidisciplinary Therapy].

A 74-year-old man visited the urology clinic with the chief complaint of urinary retention in December 2014. Serum level of initial prostate specific antigen (PSA) was 50 ng/ml and he was diagnosed with Gleason Score 4＋4 prostate adenocarcinoma with regional lymphadenopathy (cT3aN1M0). PSA level had declined after the treatment with combined androgen blockade. In November 2018, he was diagnosed with castration resistant prostate cancer (CRPC) as local progression was detected by computed tomography (CT) while PSA level did not increase. Since local symptoms worsened, resulting in repeated hematuria after the treatment with enzalutamide, palliative radiation therapy to the prostate (45 Gy) was performed. Five months later, follow-up CT showed multiple metastasis in bilateral lung and left testicle. Serum level of neuron-specific enolase (NSE) was 24.4 ng/ml without an elevated in serum PSA level. He received rebiopsy of the prostate, but no malignant findings were observed. Consequently, bilateral orchiectomy was performed for diagnosis of left testicular tumor. Pathological examination revealed metastasis of neuroendocrine prostate cancer (NEPC). Chemotherapy using cisplatin and irinotecan was administered after orchiectomy. Complete response of lung lesions was achieved and serum level of NSE decreased within normal range. No recurrence has been confirmed for 4 years after the completion of chemotherapy.

Populus alba cationic cell-wall-bound peroxidase (CWPO-C) regulates the plant growth and affects auxin concentration in Arabidopsis thaliana.

The poplar cationic cell-wall-bound peroxidase (CWPO-C) mediates the oxidative polymerization of lignin precursors, especially sinapyl alcohols, and high molecular weight compounds that cannot be oxidized by other plant peroxidases, including horseradish peroxidase C. Therefore, CWPO-C is believed to be a lignification-specific peroxidase, but direct evidence of its function is lacking. Thus, the CWPO-C expression pattern in Arabidopsis thaliana (Arabidopsis) was determined using the ß-glucuronidase gene as a reporter. Our data indicated that CWPO-C  was expressed in young organs, including the meristem, leaf, root, flower, and young xylem in the upper part of the stem. Compared with the wild-type control, transgenic Arabidopsis plants overexpressing CWPO-C had shorter stems. Approximately 60% of the plants in the transgenic line with the highest CWPO-C content had curled stems. These results indicate that CWPO-C plays a role in cell elongation. When plants were placed horizontally, induced CWPO-C expression was detected in the curved part of the stem during the gravitropic response. The stem curvature associated with gravitropism is controlled by auxin localization. The time needed for Arabidopsis plants overexpressing CWPO-C placed horizontally to bend by 90° was almost double the time required for the similarly treated wild-type controls. Moreover, the auxin content was significantly lower in the CWPO-C-overexpressing plants than in the wild-type plants. These results strongly suggest that CWPO-C has pleiotropic effects on plant growth and indole-3-acetic acid (IAA) accumulation. These effects may be mediated by altered IAA concentration due to oxidation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01241-0.

Correction to: Populus alba cationic cell-wall-bound peroxidase (CWPO-C) regulates the plant growth and affects auxin concentration in Arabidopsis thaliana.

[This corrects the article DOI: 10.1007/s12298-022-01241-0.].

Identification of Enzymes from Pseudogluconobacter saccharoketogenes Producing d-Glucaric Acid from d-Glucose.

In 2004, the US Department of Energy listed d-glucaric acid as one of the top 12 bio-based chemicals and a potential biopolymer building block. In this study, we show that Pseudogluconobacter saccharoketogenes strains can produce d-glucaric acid from d-glucose, although in low yield because of the generation of the byproduct 2-keto-d-gluconic acid in large quantities. To improve d-glucaric acid yield, we generated Rh47-3, a P. saccharoketogenes IFO14464 mutant, which produced d-glucaric acid from d-gluconic acid and d-glucose with 81 and 53 mol% yields, respectively. Furthermore, the key enzymes involved in d-glucaric acid production, alcohol dehydrogenase (Ps-ADH), aldehyde dehydrogenase (Ps-ALDH), and gluconate 2-dehydrogenase (Ps-GADH), were purified and their roles in d-glucaric acid synthesis were evaluated. Ps-ADH and Ps-ALDH catalyzed d-glucaric acid production, which was mediated by d-gluconic acid and d-glucuronic acid pathways. In contrast, Ps-GADH inhibited d-glucaric acid production by promoting the formation of 2-keto-d-gluconic acid from d-glucose.

PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces.

BACKGROUND: Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. RESULTS: We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. CONCLUSIONS: These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Targeting Ovarian Cancer Cells Overexpressing CD44 with Immunoliposomes Encapsulating Glycosylated Paclitaxel.

Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC50) for gPTX range from 15⁻20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Airborne Monoterpenes Emitted from a Cupressus lusitanica Cell Culture Induce a Signaling Cascade that Produces ß-Thujaplicin.

A cell culture of Cupressus lusitanica was used to investigate the reaction of a plant to certain airborne chemicals. Compared with laboratory and field methods using intact plants or tissues, a cell culture is advantageous because it is not affected by environmental factors, and the experiments are easier to reproduce. When exposed to an elicitor, our cell line produces 10 monoterpenes and ß-thujaplicin, which is a strong phytoalexin. These monoterpenes are emitted into the vapor phase and are expected to play a role in airborne signaling. In the present study, the cells were exposed to monoterpene vapors, and the volatiles present in the culture flasks were monitored. When the culture cells were exposed to low doses of sabinene, we detected γ-terpinene and p-cymene. After exposure to γ-terpinene, we found p-cymene and terpinolene, whereas p-cymene exposure resulted in terpinolene emission. By contrast, the other seven monoterpenes we investigated did not induce any emissions of other monoterpenes. These results strongly suggest that in C. lusitanica a signaling cascade exists that starts with the emission of sabinene and moves to γ-terpinene, p-cymene, and finally to terpinolene, which accelerates the production of the phytoalexin ß-thujaplicin.

Simultaneously disrupting AtPrx2, AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem.

Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, AtPrx2, AtPrx25 and AtPrx71, are involved in stem lignification using T-DNA insertion mutants, atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71, and investigated the impact of the simultaneous deficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerol-ß-aryl (ß-O-4) linkage type, especially ß-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants exhibited severe growth defects, such as shorter plant stature, dwarfing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass.

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 ß-fructofuranosidase.

A ß-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-ß-D-galactosylsucrose). Thermostability of the ß-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

Antitermite activity of beta-caryophyllene epoxide and episulfide.

Caryophyllene-6,7-epoxide and caryophyllene-6,7-episulfide can be easily synthesized from beta-caryophyllene by autoxidation or episulfidation. The bioactivities of beta-caryophyllene and its derivatives were investigated against the subterranean termite Reticulitermes speratus Kolbe. The antifeedant, feeding, and termiticidal activities of each compound were tested using no-choice, dual-choice, and non-contact methods. Antitermitic activities were not shown by beta-caryophyllene, but were observed for the oxide and sulfide derivatives. Caryophyllene-6,7-episulfide showed especially high antifeedant and termiticidal activities. Thus, naturally abundant, non-bioactive beta-caryophyllene can be easily converted into an antitermite reagent via a non-biological process.

Identification of Pathways for Production of D-Glucaric Acid by Pseudogluconobacter saccharoketogenes.

Pseudogluconobacter saccharoketogenes produces glucaric acid from D-glucose via two pathways, i.e., through D-glucuronic acid or D-gluconic acid. These pathways are catalyzed by alcohol dehydrogenase, aldehyde dehydrogenase, and gluconate dehydrogenase. Although D-glucaraldehyde and L-guluronic acid are also theorized to be produced in pathways throsugh D-glucuronic acid and D-gluconic acid, respectively, no direct data to identify these intermediates have been reported. In this study, the intermediates were purified and identified as D-glucaraldehyde and L-guluronic acid. The substrate specificities of the three enzymes on these intermediates and their oxidation products were studied, and the roles of alcohol, aldehyde, and gluconate dehydrogenases in D-glucaric acid-producing pathways were elucidated using the intermediates. Additionally, the substrate specificities of alcohol and aldehyde dehydrogenases on some alcohols, aldehydes, and aldoses were determined. Alcohol dehydrogenase showed wide substrate specificities, whereas the substrates oxidized by aldehyde dehydrogenase were limited. A 30-L scale reaction using the resting cells of Rh47-3 revealed that D-glucaric acid was produced from D-glucose and D-gluconic acid in 60.3 mol% (7.0 g/L) and 78.6 mol% (22.5 g/L) yields, respectively.

Alpha-linked galactooligosaccharide suppresses small intestinal eosinophil infiltration and improves growth performance in weaning pigs.

A healthy pig shows significant eosinophil infiltration in the lamina propria of the small intestine. Although the exact role of these infiltrated eosinophils in healthy pigs is unclear, eosinophil infiltration is a well-known phenomenon that is frequently observed during an allergic status. Alpha-linked galactooligosaccharide (GOS) reduces eosinophil infiltration into broncho-alveolar lavage fluid of an allergic airway eosinophilia model. We evaluated the effect of GOS oral administration on the suppression of eosinophil infiltration in the small intestine of healthy weaned pigs. Nine 21-day-old pigs were purchased and divided into three groups. One group was fed the basal diet supplemented with sucrose at 0.11% (C), one group was fed the basal diet supplemented with GOS at 1.17% (GOS A) and one group was fed the basal diet supplemented with GOS at 0.03% (GOS B). Each group was fed its respective diet throughout this study (10 days). The daily body weight gain from d3 to d10 was significantly bigger in the GOS B group than in the other groups. The feed conversion ratios from d0 to d10 were two times lower in the GOS B group than in the C group. Dietary GOS suppressed eosinophil infiltration in the small intestine. However, GOS administration had no effect on the organic acid level or microbial composition in the small and large intestinal digesta.

Complex of branched cyclodextrin and lidocaine prolonged the duration of peripheral nerve block.

Although laboratories have tried to synthesize new local anesthetics, currently available local anesthetics rarely provide prolonged regional blockade. New models of sustained-release preparations of local anesthetics with liposomes and microspheres have been studied to prolong the duration of the effects of the local anesthetics. In the present study, we examined whether a complex of a branched cyclodextrin (CD), 6-O-alpha-D-maltosyl-beta-cyclodextrin (G2-beta-CD) and lidocaine could prolong local nerve block when compared with plain lidocaine. The sciatic nerve in male Sprague-Dawley rats was blocked with plain lidocaine (n = 10), the complex of G2-beta-CD + lidocaine (n = 10), or plain G2-beta-CD (n = 4). Sensory block was assessed with a hotplate set at 56 degrees C. The median duration of the block was longer in the complex group than in the plain lidocaine group (110 min; range, 70-150 min vs 55 min; range, 40-80 min; P < 0.05), thus demonstrating that the complex with CyD significantly prolonged the nerve block effect of lidocaine. In conclusion, the present study showed that this encapsulating technique with CyD is useful to expand local anesthetic effect in peripheral nerve blockade.

Dietary alpha-linked galacto-oligosaccharide suppresses ovalbumin-induced allergic peritonitis in BALB/c mice.

To determine whether alpha-linked galacto-oligosaccharide (alpha-GOS) prevents allergic peritonitis, BALB/c mice were fed a synthetic diet with and without alpha-GOS supplementation for 7 d, and were then subcutaneously immunized with ovalbumin on days 0 and 7. The mice were challenged by intraperitoneal injection with ovalbumin on day 14, followed by peritoneal lavage on day 15. The total number of peritoneal exudate cells was significantly lower in the mice fed the alpha-GOS diet than in those fed the control diet. Peritoneal lavage fluid from mice fed the alpha-GOS diet not only had less potency to attract peripheral blood leukocytes and peritoneal exudate cells ex vivo, but also had lower concentrations of monocyte chemoattractant protein-1 (MCP-1) and eotaxin. Preincubation of the cells with alpha-GOS failed to affect the migration to peritoneal lavage fluid. We propose that dietary alpha-GOS reduces cell infiltration in allergic peritonitis by reducing antigen-induced elicitation of MCP-1 and eotaxin in mice.

Reaction mechanism of direct episulfidation of caryophyllene and humulene.

Direct episulfidations of caryophyllene or humulene with elemental sulfur were examined by means of gas chromatography. Caryophyllene-6,7-episulfide was formed at an early stage in a reaction of the caryophyllene and elemental sulfur at 120 degrees C. Caryophyllene-3,6-sulfide and polymer compounds were formed after the episulfidation. Formations of the these compounds were related to the disappearance of the caryophyllene-6,7-episulfide. Isomerization from the caryophyllene to isocaryophyllene was also observed during the reaction. In the reaction of humulene with elemental sulfur, humulene-6,7-episulfide was initially produced and then converted to humulene-9,10-episulfide. It was assumed that the polymer compound in the reaction of humulene with sulfur was related to the disappearance of the both humulene episulfides.

Thermal behavior of beta-1 subunits in lignin: pyrolysis of 1,2-diarylpropane-1,3-diol-type lignin model compounds.

1,2-Diarylpropane-1,3-diol-type lignin model compounds, 1,2-bis(4-hydroxy-3-methoxyphenyl)propane-1,3-diol (1) and 1-(3,4-diethoxyphenyl)-2-(4-methoxyphenyl)propane-1,3-diol (2), were pyrolyzed at 500 degrees C for 4 s to clarify the thermal behavior of beta-1 subunits in lignin. Products were monitored by gas chromatography/mass spectrometry. The cleavage of the Calpha-Cbeta bond to produce benzaldehydes such as 4-hydroxy-3-methoxybenzaldehyde (9) and phenylethanals as the counterparts such as 4-hydroxy-3-methoxyphenylethanal (10) occurred in pyrolyses of both 1 and 2. In pyrolysis of 1, an oxetane pathway leading to the formation of Z/E-stilbenes without the gamma-CH2OH group such as Z/E-4,4'-dihydroxy-3,3'-dimethoxystilbene (3) was predominant. In pyrolysis of 2, the oxetane pathway was minor, while pathways producing a dimer with a =CgammaH2 group by loss of water and a dimer with an alpha-carbonyl group were predominant. Pyrolysis of Japanese cedar wood provided 3 and 10 in approximately 0.8% and 0.6% yields, respectively, based on the Klason lignin content, while pyrolysis of a guaiacyl bulk dehydrogenation polymer gave them in a very small amount.

Long-term administration of 4G-beta-D-galactosylsucrose (lactosucrose) enhances intestinal calcium absorption in young women: a randomized, placebo-controlled 96-wk study.

This study determined the effect of long-term administration of 4(G)-beta-D-galactosylsucrose (lactosucrose; LS) on intestinal calcium absorption. In a randomized, single-blind, parallel-group study, LS (n=9, 6.0 g twice daily) or a placebo (maltose; n=8, 6.0 g twice daily) was administered to healthy young women for 92 wk: the study also included a 4-wk post-administration period. All participants completed the study. Dietary nutrient intake; fecal weight, pH, and moisture content; fecal concentrations of short-chain fatty acids (SCFA), putrefactive products, ammonia, and minerals (calcium, magnesium, phosphorus, and iron); and serum calcium and osteocalcin concentrations were measured every 24 wk. Urinary pyridinoline (PYR) and deoxypyridinoline (DPD), and urinary calcium excretion were measured every 12 wk. Significant effects of oligosaccharide treatment, time, and the interaction between oligosaccharide treatment and time were observed for fecal pH, SCFA, ammonia, and putrefactive product values (p<0.05). Fecal pH, ammonia, and putrefactive product values decreased in the LS group, and the fecal SCFA concentration significantly increased during the administration period; these changes were not observed 4 wk post-administration. To examine the mineral balance of calcium, magnesium, and phosphorus in detail, all the participants completed a 6-d mineral balance study, sometime between week 56 and 60 of the longer study. During the mineral balance study, the daily calcium intake was set at 400 mg; all feces and urine were collected each day for 6 d after an 8-d acclimation period. In the balance study, fecal calcium excretion was significantly lower in the LS group than in the placebo group (p<0.05), and apparent calcium absorption and retention, apparent magnesium and phosphorus absorption, and magnesium retention were significantly higher in the LS group than in the placebo group (p<0.05). Our results suggest that the administration of LS produces a long-term enhancement of intestinal calcium absorption in healthy young women with lower than recommended calcium intakes.

Adipocyte-specific CD1d-deficiency mitigates diet-induced obesity and insulin resistance in mice.

It has been shown that CD1d expression and glycolipid-reactive, CD1d-restricted NKT cells exacerbate the development of obesity and insulin resistance in mice. However, the relevant CD1d-expressing cells that influence the effects of NKT cells on the progression of obesity remain incompletely defined. In this study, we have demonstrated that 3T3-L1 adipocytes can present endogenous ligands to NKT cells, leading to IFN-γ production, which in turn, stimulated 3T3-L1 adipocytes to enhance expression of CD1d and CCL2, and decrease expression of adiponectin. Furthermore, adipocyte-specific CD1d deletion decreased the size of the visceral adipose tissue mass and enhanced insulin sensitivity in mice fed a high-fat diet (HFD). Accordingly, NKT cells were less activated, IFN-γ production was significantly reduced, and levels of adiponectin were increased in these animals as compared with control mice on HFD. Importantly, macrophage recruitment into the adipose tissue of adipocyte-specific CD1d-deficient mice was significantly blunted. These findings indicate that interactions between NKT cells and CD1d-expressing adipocytes producing endogenous NKT cell ligands play a critical role in the induction of inflammation and functional modulation of adipose tissue that leads to obesity.