A Convenient Synthesis Method for Methylenomycin B and Its Application to Methylenomycin A

A convenient synthesis method for methylenomycin B and its homolog, methylenomycin A, has been developed. Methylenomycin B, 2,3-dimethyl-5-methylene-2-cyclopentenone (3) was synthesized: i) by methylation of Mannich derivative prepared from morpholine and 2,3-dimethyl- 2-cyclopentenone (5) or ii) by treatment of formalin with sodio derivatives of 2,3-dimethyl-5-formyl-2-cyclopentenone (7a) and 2,3-dimethyl-5-ethoxalyl-2-cyclopentenone (7b), both of which were easily prepared from 5 and ethyl formate or ethyl oxalate. 2,3-Dimethyl-2,3-epoxy-5-methylenecyclopentanone (2) was similarly prepared from the epoxide compound of 5 and ethyl oxalate. The bioassay ofmethylenomycin B and its related compounds against bacteria (B. subtilis, S. aureus, Ps. aeruginosa and E. coli) was also conducted. Methylenomycin A (1) and its desepoxy compound (17) were also prepared from 4-carboxy-2,3-dimethyl-2-cyclopentenone (15) in the same procedure as described above.     

Synthesis of 2-Hydroxy-3-p-tolyl-2-cyclopentenone and Its Application to Isolaurene

2-Hydroxy-3-p-tolyl-2-cyclopentenone (7), a potential starting material for synthesis of isolaurene (1), was prepared by photolysis of 2-p-toluenesulfonyloxy-2-cyclopentenone (6). Conversion of 7 into 2,5-dimethyl-2-p-tolyl-cyclopentanone (14) was carried out as follows. Methylation of 7 with methyl iodide gave 2-methoxy-3-p-tolyl-2-cyclopentanone (9), and the treatment of (9) with methyl magnesium iodide afforded 2-methyl-5-p-tolyl-2-cyclopentenone (11). This compound was allowed to react with methyl iodide in the presence of sodium methoxide to yield 2,5-dimethyl-2-p-tolyI-4-cyclopentenone (13), the hydrogenation of which over palladium charcoal gave 14.     

Evolutionary Changes in Vertebrate Genome Signatures with Special Focus on Coelacanth

With a remarkable increase in genomic sequence data of a wide range of species, novel tools are needed for comprehensive analyses of the big sequence data. Self-organizing map (SOM) is a powerful tool for clustering high-dimensional data on one plane. For oligonucleotide compositions handled as high-dimensional data, we have previously modified the conventional SOM for genome informatics: BLSOM. In the present study, we constructed BLSOMs for oligonucleotide compositions in fragment sequences (e.g. 100 kb) from a wide range of vertebrates, including coelacanth, and found that the sequences were clustered primarily according to species without species information. As one of the nearest living relatives of tetrapod ancestors, coelacanth is believed to provide access to the phenotypic and genomic transitions leading to the emergence of tetrapods. The characteristic oligonucleotide composition found for coelacanth was connected with the lowest dinucleotide CG occurrence (i.e. the highest CG suppression) among fishes, which was rather equivalent to that of tetrapods. This evident CG suppression in coelacanth should reflect molecular evolutionary processes of epigenetic systems including DNA methylation during vertebrate evolution. Sequence of a de novo DNA methylase (Dntm3a) of coelacanth was found to be more closely related to that of tetrapods than that of other fishes.     

Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine

We have established a manufacturing system for a Vero cell-derived inactivated Japanese encephalitis vaccine at a 500l scale. The production system involves expansion of Vero cells using microcarrier, followed by virus infection. Except for an additional purification step, the downstream purification processes are similar to those used for the current mouse brain-derived vaccine; cell removal, concentration and removal of low-molecular weight impurities by membrane filtration, formalin-inactivation, sucrose density gradient ultracentrifugation, and Sulfate-Cellulofine column chromatography are conducted. The antigen obtained from the manufacturing system was highly purified and its physico-chemical and immunological properties were comparable with those of antigen derived from mouse brains. Our system is very simple and could be easily scaled-up to allow vaccine production at a several thousand litre scale.     

An Efficient Synthetic Method for (±)-Malyngolide and Its Optical Resolution

A convenient synthetic method for (±)-malyngolide (1), an antibiotic from the marine bluegreen alga Lyngbya majuscula GOMONT, is described. The compound (±)-1 was synthesized from 2-methylglutaric anhydride (starting material) in four steps, involving the TBHP-based osmium-catalyzed procedure for vicinal dihydroxylation of the methylene unit in unsaturated carboxylic acid as the key step. The optical resolution of (±)-l was accomplished by HPLC, in which diastereomeric (+)-2-(4-chlorophenyl)isovaleric acid [(+)-CPIA] esters of (±)-l derived from (+)-CPIA-C1 could be easily separated, and subsequent hydrolysis of each ester gave the enantiomeri-cally pure (?)-l and (+)-l, respectively. A synthetic method for α-methylene malyngolide, which is to be expected for biological activity, is also described.     

Effect of One-Week Salt Restriction on Blood Pressure Variability in Hypertensive Patients with Type 2 Diabetes

Background

Increased short-term blood pressure (BP) variability on 24-hour ambulatory BP monitoring (ABPM) is known to be a risk factor for cardiovascular events. However, very few studies have evaluated the effect of salt restriction on BP variability particularly in hypertensive patients with type 2 diabetes. This study aimed to investigate the effect of salt restriction on systolic BP (SBP) variability.

Methods and Results

10 hypertensive patients with type 2 diabetes and not receiving antihypertensive agents were enrolled in the study. After admission, all patients received a salt-restricted diet and appropriate anti-diabetic treatments and were followed up for 7 consecutive days using ABPM. After the 7-day treatment, the median [interquartile range (IQR)] coefficient of variation (CV) for diurnal SBP variability changed from day 1 to day 7–13.0 [10.8 to 16.8] % to 13.3 [9.1 to 18.9] % (P = 0.959)—and the median [IQR] change between days 1 and 7 was -0.3 [-3.2 to 2.9] %. In addition, CV for BP variability and circadian rhythm of BP varied greatly on a day-by-day basis for 7 days, compared to mean BP values. Interestingly, increased SBP variability was associated with greater day-by-day changes in circadian rhythm of BP.

Conclusions

Salt restriction during 7-day hospitalization led to a -0.3 [-3.2 to 2.9] (median [IQR]) % change from baseline in CV for diurnal SBP variability in 10 hypertensive patients with type 2 diabetes not receiving antihypertensive agents.

Trial Registration

UMIN Clinical Trials Registry UMIN000016243     

Changes in Expression of Aquaporin-4 and Aquaporin-9 in Optic Nerve after Crushing in Rats

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.     

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm² (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm² (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm² (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.