Metathesis-mediated synthesis of (R)-10-methyl-2-tridecanone, the southern corn rootworm pheromone

(R)-10-Methyl-2-tridecanone, the female sex pheromone of the southern corn rootworm (Diabrotica undecimpunctata howardi Barber), was synthesized in 9 steps from methyl (S)-3-hydroxy-2-methylpropanoate in a 15.7% overall yield. Olefin cross metathesis between (R)-6-methyl-1-nonene and 5-hexen-2-one employing Grubbs' first-generation catalyst was the key step of the synthesis.     

Maximal voluntary tongue pressure is decreased in Japanese frail elderly persons

doi: 10.1111/j.1741‐2358.2011.00615.x Maximal voluntary tongue pressure is decreased in Japanese frail elderly persons Background and objective: To quantitatively estimate tongue function, we developed a handy device for intraoral pressure measurement. The objective of this study was to assess maximum voluntary tongue pressure (MVTP) in Japanese frail elderly persons receiving nursing care services. Materials and methods: The study included 42 men and 87 women, aged 58–100 years. To record MVTP, the participants were asked to compress the balloon (diameter: 18 mm) of the disposable intraoral pressure probe onto their palates for 7 s using the maximum voluntary effort of the tongue. Pressures were recorded three times at 1 min intervals. Results: Maximum voluntary tongue pressure was successfully measured in 111 persons. Mean (standard deviation) MVTP was 18 (12) kPa, with a range of 0–63 kPa. The remaining 18 persons could not accurately follow our instructions and MVTP could not be measured. Conclusion: In comparison with the reported standard value using the same device, the frail elderly persons included in our study were found to exert less MVTP than healthy dentate individuals. These results suggest the need for proper quantitative evaluation of oral function, including tongue capacity, at nursing care facilities.     

Tail variation of the folding primer affects the SmartAmp2 process differently

Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either “strong” or “weak”, depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with “strong tails”. Unlike “strong tails”, concentration change of FP with “weak tails” did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.     

Sodium hydroxide based non-detergent decellularizing solution for rat lung

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.     

Ceramide Induces Apoptosis to Immature Cerebellar Granule Cells in Culture

A recent study revealed that ceramide acts as a second messenger in the sphingomyelin pathway and thus plays an important regulatory role in programmed cell death (apoptosis) to cell the lines induced by tumor-necrosis factor (TNF)- and interleukin (IL)-1, although its effect remains controversial regarding primary neuronal culture. We investigated the effect of a cell-permeable ceramide analog (C₂-ceramide) on cultures of cerebellar granule cells, which is thought to have active sphingomyelin pathway during development. The presence of C₂-ceramide decreased the number of cerebellar granule cells (CGCs) in a concentration-dependent manner when added at DIV 1 (1 day in vitro). The ED₅₀ was 60 M. After DIV2, CGCs became less sensitive to C₂-ceramide and the ED₅₀ was 200 M at DIV 7. DNA staining with Hoechst 33258 showed the morphology of apoptotic nuclei in the degenerating neurons. Internucleosomal DNA degradation could also be observed by gel electrophoresis. Protein and RNA synthesis inhibitors prevented the death of neurons. C₂-dihydroceramide, which lacks the 4–5 trans double bond and failed to induce neuronal death. These results thus demonstrated that C₂-ceramide induces apoptosis to the CGCs at the early stage in vitro, however the CGCs were found to be less sensitive to C₂-ceramide at the later stage in vitro.     

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.     

sigma marY1, the LTR of the gypsy-type retroelement marY1 from the basidiomycete tricholoma matsutake, allows multicopy DNA integration in Lentinula edodes

sigma marY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a sigma marY1 derivative, sigma* marY1, conferred the hygromycin-resistant phenotype stronger than the vector without sigma* marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that sigma marY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.     

Immunotherapy and combined assay of serum levels of carcinoembryonic antigen and acute-phase reactants

 Our previous studies have revealed that gastric and esophageal cancer patients with abnormal sialic acid levels had a better response than those with normal levels if they received polysaccharide K (PSK), a nonspecific immunomodulator. Serum levels of carcinoembryonic antigen (CEA) and acute-phase reactants (APR) such as immunosuppressive acidic protein, acid-soluble glycoproteins, α1-antichymotrypsin, and sialic acid were analyzed in 872 gastric cancer patients who had undergone resection from March 1979 to September 1993 at the Department of Surgery of Tokai University. The patients were categorized into four groups according to the preoperative serum levels: group A had normal levels of both CEA and APR, group B had abnormal CEA and normal APR levels, group C had a normal CEA level and normal levels of one or more APR, and group D had abnormal levels of both CEA and of one or more APR. Patients in group D who received PSK showed significantly better survival than those without PSK (29.3% versus 6.9%; log-rank test, P = 0.0015; Breslow test, P = 0.0042). CEA-positive patients receiving PSK therapy exhibited a significantly better survival rate than those without PSK (38.1% versus 18.6%; log-rank test, P = 0.0136; Breslow test, P = 0.0125). Cox’s regression analysis showed that PSK therapy was significantly related to survival in group D, but not in the other groups. We conclude that the combined assay of tumor-associated factors (such as CEA) and various nonspecific reactants to the presence of cancer (such as immunosuppressive acidic protein, α1-antichymotrypsin, acid-soluble glycoproteins and sialic acid) provides a good set of preoperative indicators on which to base the selection of treatment for individual gastric cancer patients. Received: 25 July 1997 / Accepted: 5 November 1997     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells

Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O₂ permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactae production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements. Correspondence to: M. Ogata