Openable artificial intestinal tract device integrated with a permeable filter for evaluating drug permeation through cells.

Organs-on-chips using cultured cells have been developed and applied for evaluating in vitro biological phenomena. We previously reported an openable artificial intestinal tract system, as an in vitro model of the small intestine, for in vitro drug screening. The intestinal tract device could be transformed using an integrated artificial muscle actuator. An initial flat state was suitable for cell culture, and the transformed tubular structure was used as a fluidic channel for perfusion tests. The previously developed intestinal tract system could be used to evaluate drug absorption by cells through perfusion testing. This study presents an improved artificial intestinal tract system for analysis of drug permeation, in addition to absorption. Permeable filters were integrated into the intestinal tract device. Integration of additional filters into the design of the existing artificial muscle actuator was accomplished by considering device performance and available filter locations. Filter permeability was evaluated by perfusion testing. MDCK-II cells were cultured on the device and visually and electrically evaluated. The openable device, equipped with new functions for further pharmacokinetic analysis, could perform and evaluate drug disposition using cultured cells. We anticipate that the improved, openable organ-on-a-chip device system will contribute to advances in in vitro drug screening technology.

Modulatory effects of repeated psychophysical stress on masseter muscle nociception in the nucleus raphe magnus of rats.

Psychophysical stress can cause neural changes that increase nociception in the orofacial region, particularly the masseter muscle (MM). The nucleus raphe magnus (NRM), which is located in the brain stem, serves the crucial role of regulating nociception through descending modulatory pain control. However, it remains unclear if neural activities in the NRM are affected under psychophysical stress conditions. This study conducted experiments to assess (1) whether neural activity, indicated by Fos expression in an NRM that has experienced MM injury, is affected by the stress of repeated forced swim tests (FST); and (2) whether the selective serotonin reuptake inhibitor fluoxetine administered daily after an FST could affect the number of Fos-positive neurons in the NRM. Results revealed that the stress from repeated FSTs significantly increased the number of Fos-positive neurons in an NRM that had been affected by MM injury. Fluoxetine inhibited increases in the number of Fos-positive neurons in the NRM that occurred as a result of FSTs, but this was not observed in sham rats. These findings indicate that the stress from FSTs could increase nociceptive neural activity in an NRM that has experienced MM injury. This could be due, in part, to changes in serotonergic mechanisms.

Daily administration of Sake Lees (Sake Kasu) reduced psychophysical stress-induced hyperalgesia and Fos responses in the lumbar spinal dorsal horn evoked by noxious stimulation to the hindpaw in the rats.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day -3 to Day -1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.

Japanese Rice Wine can reduce psychophysical stress-induced depression-like behaviors and Fos expression in the trigeminal subnucleus caudalis evoked by masseter muscle injury in the rats.

We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day -3 to -1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.

Genomic analysis of a Streptomyces tsukubaensis mutant with reduced FR900525 production isolated by selection for S-(2-aminoethyl) l-cysteine resistance.

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been shown to be of clinical importance and has been manufactured industrially since 1993 using mutants with high FK506 production ability. These mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production, and we previously established a mutant strain that produces reduced levels of FR900525 by selecting for S-(2-aminoethyl) l-cysteine (AEC) resistance. In this study, we conducted a genomic analysis of this strain to identify the changes associated with AEC resistance and to determine its metabolism. Three mutated genes were identified by comparing the genome sequences of the parental strain (A) and the AEC-resistant mutant (B). From the metabolite pathway, it was speculated that citric acid synthase was the most relevant to AEC resistance. To investigate the effect of the mutation in citric acid synthase, we added citric acid, an inhibitor of citric acid synthase, to strain A culture, which induced strain A to exhibit a strain B-like phenotype. We conclude that the mutation in citric acid synthase enhances the carbon flow into aspartic acid, increasing lysine synthesis and resulting in AEC resistance in strain B, as well as high production of FK506 and low production of FR900525.

Inhibitory effects of fluoxetine, an antidepressant drug, on masseter muscle nociception at the trigeminal subnucleus caudalis and upper cervical spinal cord regions in a rat model of psychophysical stress.

This study aimed to determine whether psychophysical stress conditionings had facilitatory effects on masseter muscle nociception in the central nervous system via serotonergic mechanisms in rats. Two experiments were conducted to assess: (1) whether repeated forced swim stress for 3 days increased the number of Fos-positive neurons evoked by masseter muscle injury due to formalin injection; and (2) whether serotonin-reuptake inhibitor, fluoxetine, administered daily after each stress conditioning, had modulatory roles on Fos expression. The number of Fos-positive cells was quantified in several areas within the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord regions (Vc areas), including the ventrolateral area of the trigeminal subnucleus interpolaris/Vc transition, and the middle or caudal portion of the Vc regions, since nociceptive neural activity in the Vc region could play critical roles in deep craniofacial nociception. We found that forced swim stress conditionings increased depression-like behaviors, which was prevented by fluoxetine. Repeated forced swim stress significantly increased Fos expression in all Vc areas compared with those of non-stressed rats, while systemic administration of fluoxetine significantly decreased Fos expression in all areas, but mainly in the caudal Vc region, in stressed rats. Fluoxetine had no effect on Fos expression in non-stressed rats. These results indicate that repeated forced swim stress conditionings increase Fos expression in the Vc areas, and the contribution of serotonergic mechanisms to masseter muscle nociception could be greater in stressed rats than in sham rats. These results support the hypothesis that changes in brain function, including serotonergic mechanisms, in the Vc areas play critical roles in enhanced masseter muscle nociceptive responses under psychophysical stress conditions.

Strain improvement of Lentzea sp. 7887 for higher yield per unit volume on hydroxylation of cyclosporine derivative FR901459.

A Gram-positive bacterium Lentzea sp. 7887 hydroxylates a cyclosporine derivative FR901459 into AS1837812 (9-hydroxide), which is an important intermediate of candidate drugs that target the hepatitis C virus. We screened a UV-induced mutant, named M-1, which showed about 1.2-fold higher conversion yields, 2-fold higher substrate concentrations (3.69 mM), and 2.5-fold higher yield per unit volume than the wild-type strain.

Reduction of FR900525 using an S-(2-aminoethyl) l-cysteine-resistant mutant.

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S. tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis.

Pilot-scale whole-cell biocatalysis for the hydroxylation of cyclosporine derivative, FR901459, at higher concentrations by Lentzea sp. 7887 using soybean flour as a novel substrate dispersant.

Pilot-scale hydroxylation of FR901459, an immunosuppressive cyclosporine derivative, was performed using resting cells of a Gram-positive bacteria Lentzea sp. 7887 (as whole-cell biocatalysts) and soybean flour as a substrate dispersant. Through biocatalysis, FR901459 was hydroxylated at position 9, producing AS1837812, an important intermediate in the production of drug candidates against hepatitis C. Since FR901459 is insoluble in water, the conversion ratio ([moles of AS1837812 produced/moles of FR901459 added]×100) of the biocatalysis decreased under conditions with substrate concentrations higher than 0.615 mM. To increase the concentration of FR901459 for biocatalysis, we screened various materials to effectively disperse FR901459 in the biocatalysis mixture and found that soybean flour was the best substrate dispersant. The addition of soybean flour to the biocatalysis mixture increased the FR901459 concentration in a 3-L reactor up to 3-fold (1.85 mM). Thus, we successfully established a pilot-scale (1-m3) biocatalysis with a 2-fold higher concentration (1.23 mM) of FR901459 using soybean flour as the substrate dispersant and obtained 419 g of AS1837812 at a conversion ratio of 34.5% in a 28-h batch reaction. Soybean flour can be used as a substrate dispersant for various industrial biocatalysis processes because of its low cost, high availability, and low environmental impact.

Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907.

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

Improvement of FR901379 production by mutant selection and medium optimization.

FR901379 (WF11899A) is a novel echinocandin type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. Micafungin (FK463) is derived from the chemical modification of deacylated FR901379. In the present paper, we performed seven generation's strain-breeding, beginning with a wild type, was performed. Selection medium for screening and production medium for high FR901379 production were designed. Sodium chloride content in the selection plate was affected to FR901379 production and shrinkage of the colony size was observed in high producing strains. As selection markers, large colony-shrinking rate and large inhibition circle in the agar-piece method using C. albicans was selected. Using CMA medium with high sodium chloride, 3 mutants, M-1 to M-3, have achieved a high FR901379 production and M-3 showed 5.0 U/mL, while 1.0 U/mL of production was achieved in wild type strain. A-2 medium supplemented with 6% of soluble starch as a carbon source and 0.6% of ammonium sulfate as nitrogen source was also further effective for mutant screening. The FR901379 production of mutant M-4 (fourth generation) increased until 16.0 U/mL. The concentration of the phosphate salt in the medium seemed to inhibit the growth so as to extend the culture period. When the A-3 medium supplemented with low concentration of phosphate salt and magnesium sulfate as a sulfate source was designed and used, mutants with improved production were successively obtained. Finally, variant strain M-7 showed 30.0 U/mL of production, which was about 30 times higher than that of the wild strain.