DNA Microarray and Bioinformatic Analysis Reveals the Potential of Whale Oil in Enhancing Hair Growth in a C57BL/6 Mice Dorsal Skin Model.

Much research has been conducted to determine how hair regeneration is regulated, as this could provide therapeutic, cosmetic, and even psychological interventions for hair loss. The current study focused on the hair growth effect and effective utilization of fatty oil obtained from Bryde's whales through a high-throughput DNA microarray approach in conjunction with immunohistochemical observations. The research also examined the mechanisms and factors involved in hair growth. In an experiment using female C57BL/6J mice, the vehicle control group (VC: propylene glycol: ethanol: water), the positive control group (MXD: 3% minoxidil), and the experimental group (WO: 20% whale oil) were topically applied to the dorsal skin of the mouse. The results showed that 3% MXD and 20% WO were more effective than VC in promoting hair growth, especially 20% WO. Furthermore, in hematoxylin and eosin-stained dorsal skin tissue, an increase in the number of hair follicles and subcutaneous tissue thickness was observed with 20% WO. Whole-genome transcriptome analysis also confirmed increases for 20% WO in filaggrin (Flg), a gene related to skin barrier function; fibroblast growth factor 21 (Fgf21), which is involved in hair follicle development; and cysteine-rich secretory protein 1 (Crisp1), a candidate gene for alopecia areata. Furthermore, the results of KEGG pathway analysis indicated that 20% WO may have lower stress and inflammatory responses than 3% MXD. Therefore, WO is expected to be a safe hair growth agent.

Transcriptomic (DNA Microarray) and Metabolome (LC-TOF-MS) Analyses of the Liver in High-Fat Diet Mice after Intranasal Administration of GALP (Galanin-like Peptide).

The aim of this research was to test the efficacy and potential clinical application of intranasal administration of galanin-like peptide (GALP) as an anti-obesity treatment under the hypothesis that GALP prevents obesity in mice fed a high-fat diet (HFD). Focusing on the mechanism of regulation of lipid metabolism in peripheral tissues via the autonomic nervous system, we confirmed that, compared with a control (saline), intranasally administered GALP prevented further body weight gain in diet-induced obesity (DIO) mice with continued access to an HFD. Using an omics-based approach, we identified several genes and metabolites in the liver tissue of DIO mice that were altered by the administration of intranasal GALP. We used whole-genome DNA microarray and metabolomics analyses to determine the anti-obesity effects of intranasal GALP in DIO mice fed an HFD. Transcriptomic profiling revealed the upregulation of flavin-containing dimethylaniline monooxygenase 3 (Fmo3), metallothionein 1 and 2 (Mt1 and Mt2, respectively), and the Aldh1a3, Defa3, and Defa20 genes. Analysis using the DAVID tool showed that intranasal GALP enhanced gene expression related to fatty acid elongation and unsaturated fatty acid synthesis and downregulated gene expression related to lipid and cholesterol synthesis, fat absorption, bile uptake, and excretion. Metabolite analysis revealed increased levels of coenzyme Q10 and oleoylethanolamide in the liver tissue, increased levels of deoxycholic acid (DCA) and taurocholic acid (TCA) in the bile acids, increased levels of taurochenodeoxycholic acid (TCDCA), and decreased levels of ursodeoxycholic acid (UDCA). In conclusion, intranasal GALP administration alleviated weight gain in obese mice fed an HFD via mechanisms involving antioxidant, anti-inflammatory, and fatty acid metabolism effects and genetic alterations. The gene expression data are publicly available at NCBI GSE243376.

Docosapentaenoic acid-rich oil lowers plasma glucose and lipids in a mouse model of diabetes and mild obesity.

Many studies have investigated the beneficial effects of n-3 polyunsaturated fatty acids, such as their potential for lowering lipid levels and reducing diabetes risk. However, few studies have specifically examined docosapentaenoic acid (DPA), an n-3 polyunsaturated fatty acid with limited availability in its pure form. We hypothesized that DPA would have lipid-lowering effects and improve insulin resistance in KK/Ta mice. To test our hypothesis, 7-week-old KK/Ta mice were fed a high-fat diet for 12 weeks to induce obesity before being divided into 3 groups and fed an experimental diet for 10 weeks. The experimental diets were: LSO, using lard and safflower oil as fat sources; SO, in which lard in the LSO diet was replaced with safflower oil; and DPA, in which lard in the LSO diet was replaced with DPA oil. After 10 weeks, plasma triglyceride and total cholesterol concentrations were significantly decreased in the DPA group, but not in the SO group. Sterol regulatory element-binding protein-1 and stearoyl-CoA desaturase-1 gene expressions involved in fatty acid synthesis in the liver were significantly lower in the DPA group compared with the LSO group. Plasma glucose concentrations were significantly decreased in both the SO group and the DPA group compared with the LSO group, whereas plasma insulin concentrations were significantly decreased in the DPA group alone. These results indicate that DPA has plasma lipid-lowering and hypoglycemic effects, possibly from suppression of fatty acid synthesis in the liver.

Altered renin-angiotensin system gene expression in airways of antigen-challenged mice: ACE2 downregulation and unexpected increase in angiotensin 1-7.

OBJECTIVE: Evidence suggest that the renin-angiotensin system (RAS) is activated in people with asthma, although its pathophysiological role is unclear. Angiotensin-converting enzyme 2 (ACE2) is the major enzyme that converts angiotensin II to angiotensin 1-7 (Ang-1-7), and is also known as a receptor of SARS-CoV-2. The current study was conducted to identify the change in RAS-related gene expression in airways of a murine asthma model. METHODS: The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. RESULTS: The KEGG pathway analysis of differentially expressed genes in our published microarray data revealed a significant change in the RAS pathway in the antigen-challenged mice. Quantitative RT-PCR analyses showed significant increases in the angiotensin II-generating enzymes (Klk1, Klk1b3 and Klk1b8) and a significant decrease in Ace2. Surprisingly, ELISA analyses revealed a significant increase in Ang-1-7 levels in bronchoalveolar lavage (BAL) fluids of the antigen-challenged animals, while no significant change in angiotensin II was observed. Application of Ang-1-7 to the isolated BSMs had no effect on their isometrical tension. CONCLUSION: The expression of Ace2 was downregulated in the BSMs of OA-challenged mice, while Klk1, Klk1b3 and Klk1b8 were upregulated. Despite the downregulation of ACE2, the level of its enzymatic product, Ang-1-7, was increased in the inflamed airways, suggesting the existence of an unknown ACE2-independent pathway for Ang-1-7 production. The functional role of Ang-1-7 in the airways remains unclear.

Molecular and Physiological Functions of PACAP in Sweat Secretion.

Sweat plays a critical role in human body, including thermoregulation and the maintenance of the skin environment and health. Hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion, resulting in severe skin conditions (pruritus and erythema). Bioactive peptide and pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and identified to activate adenylate cyclase in pituitary cells. Recently, it was reported that PACAP increases sweat secretion via PAC1R in mice and promotes the translocation of AQP5 to the cell membrane through increasing intracellular [Ca2+] via PAC1R in NCL-SG3 cells. However, intracellular signaling mechanisms by PACAP are poorly clarified. Here, we used PAC1R knockout (KO) mice and wild-type (WT) mice to observe changes in AQP5 localization and gene expression in sweat glands by PACAP treatment. Immunohistochemistry revealed that PACAP promoted the translocation of AQP5 to the lumen side in the eccrine gland via PAC1R. Furthermore, PACAP up-regulated the expression of genes (Ptgs2, Kcnn2, Cacna1s) involved in sweat secretion in WT mice. Moreover, PACAP treatment was found to down-regulate the Chrna1 gene expression in PAC1R KO mice. These genes were found to be involved in multiple pathways related to sweating. Our data provide a solid basis for future research initiatives in order to develop new therapies to treat sweating disorders.

Ontogenetic development of the water channel protein AQP5 in mouse salivary gland tissue.

Aquaporins (AQP) are a family of channel proteins expressed in the cell membranes of many tissue types. As water channels, they enable the selective permeation of water molecules and thus play an important role in water transport through the plasma membrane. There are numerous AQP sub-types, among which AQP5 is expressed in the salivary glands. The expression and localization of AQP5 in different salivary gland cells of animal models during fetal development and after birth have enabled the physiological functions of AQP5 to be elucidated, but subsequent changes in the adult phase are unknown. It is known that saliva production tends to decrease with age, but it is unclear how AQP5 activity and function changes developmentally, from young to old including gender differences. In the present study, we sampled the parotid, submandibular, and sublingual glands from young (8 weeks old) and aged (12 months old) mice of both sexes to study the effects of age- and sex-related differences in AQP5 expression. Positive fluorescence immunostaining was detected in the membranes of cells from all gland types, and this was enhanced in juvenile mice from both sexes. Western blot analyses revealed that AQP5 expression levels tended to decrease with age in both male and female animals. Conversely, AQP5 gene expression levels did not change significantly with aging, but were found to be high in submandibular gland cells of both sexes, in parotid gland cells of older female mice, and in the sublingual gland cells of young male mice.

Examining the Effect of Notocactus ottonis Cold Vacuum Isolated Plant Cell Extract on Hair Growth in C57BL/6 Mice Using a Combination of Physiological and OMICS Analyses.

The biological and psychological importance of hair is recognized worldwide. Molecules that can promote the activation of hair follicle stem cells and the initiation of the growth phase have been subjects of research. Clarifying how hair regeneration is regulated may help to provide hair loss treatments, including cosmetic and even psychological interventions. We examined the hair-growing effects of a cell extract (CE) obtained from cactus Notocactus ottonis by the cold vacuum extraction protocol, by investigating its hair-growing effects, relevant mechanisms, and potential factors therein. Using male C57BL/6 mice, vehicle control (VC: propylene glycol: ethanol: water), MXD (minoxidil, positive control), and N. ottonis CE (N-CE, experimental) were applied topically to the backs of mice. The results showed that MXD and N-CE were more effective in promoting hair growth than VC. An increase in number of hair follicles was observed with N-CE in hematoxylin-eosin-stained skin tissue. The metabolite composition of N-CE revealed the presence of growth-promoting factors. Using mouse back whole-skin tissue samples, whole-genome DNA microarray (4 × 44 K, Agilent) and proteomics (TMT-based liquid chromatography-tandem mass spectrometry) analyses were carried out, suggesting the molecular factors underlying hair-promoting effects of N-CE. This study raises the possibility of using the newly described N. ottonis CE as a hair-growth-promoting agent.

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3ß, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

Potential Therapeutic Role of Pituitary Adenylate Cyclase-Activating Polypeptide for Dry Eye Disease.

Dry eye disease (DED) is caused by a reduction in the volume or quality of tears. The prevalence of DED is estimated to be 100 million in the developed world. As aging is a risk factor for DED, the prevalence of DED is expected to grow at a rapid pace in aging populations, thus creating an increased need for new therapies. This review summarizes DED medications currently in clinical use. Most current medications for DED focus on stimulating tear secretion, mucin secretion, or suppressing inflammation, rather than simply replenishing the ocular surface with moisture to improve symptoms. We recently reported that the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) induces tear secretion and suppresses corneal injury caused by a reduction in tears. Moreover, it has been reported that a PACAP in water and a 0.9% saline solution at +4 °C showed high stability and achieved 80-90% effectiveness after 2 weeks of treatment. These results reveal PACAP as a candidate DED medication. Further research on the clinical applications of PACAP in DED is necessary.

Effect of PACAP on sweat secretion by immortalized human sweat gland cells.

The process of sweating plays an important role in the human body, including thermoregulation and maintenance of the environment and health of the skin. It is known that the conditions of hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion and can result in severe skin conditions such as pruritus and erythema, which significantly reduce the patient's quality of life. However, there are many aspects of the signaling mechanisms in the process of sweating that have not been clarified, and no effective therapies or therapeutic agents have yet been discovered. Previously, it was reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes sweating, but details of the underlying mechanism has not been clarified. We used immortalized human eccrine gland cells (NCL-SG3 cell) to investigate how sweat secretion is induced by PACAP. Intracellular Ca2+ levels were increased in these cells following their exposure to physiological concentrations of PACAP. Intracellular Ca2+ was not elevated when cells were concomitantly treated with PA-8, a specific PAC1-R antagonist, suggesting that PAC1-R is involved in the elevation of intracellular Ca2+ levels in response to PACAP treatment. Furthermore, immunocytochemistry experiments showed that aquaporin-5 was translocated from the cytoplasm to the cell membrane by PACAP. These results suggest that PACAP acts on eccrine sweat glands to promote sweat secretion by translocation of aquaporin-5 to the cell membrane in response to increased levels of intracellular Ca2+. These findings also provide a solid basis for future research initiatives to develop new therapies to treat sweating disorders.

Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line-PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3ß (GSK-3ß) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3ß as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3ß inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10-7 M, 10-8 M, and 10-9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10-7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3ß-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

Exploring natural microbial resources for the discovery of anti-malarial compounds.

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

Analgesic effect of mineral cream containing natural spa minerals for use on the skin.

Previous studies have shown that dissolved substances in some natural hot springs have analgesic/anti-nociceptive and anti-inflammatory actions. However, the mechanisms underlying how such dissolved substances exert these actions are not fully understood. In the present study on mice, we examined the analgesic/anti-nociceptive and anti-inflammatory properties of a mineral cream containing natural hot spring ingredients. The anti-nociceptive effects of the mineral cream were assessed by using the von Frey test. Application of the mineral cream to the hind paw of mice produced a significant anti-nociceptive effect compared to control. The anti-nociceptive effects of the mineral cream were also assessed following the injection of complete Freund's adjuvant (CFA) into the hind paws of mice after pre-treatment for one or four weeks with the mineral cream. Histological experiments with light microscopy showed that the mineral cream did not reduce inflammation caused by the CFA treatment. In addition, the mineral cream did not inhibit oxidative stress as evidenced by increased levels of oxidative metabolites (d-ROMs) and biological anti-oxidant potential (BAP). These results suggest that the mineral cream does not exert a protective effect against inflammation, and that the constituents of the mineral cream may produce their anti-nociceptive effects transdermally via different mechanisms including the nervous system.

Cloning and expression of the FR901379 acylase gene from Streptomyces sp. no. 6907.

FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80 kDa, two-subunit heterodimeric protein characteristic of the ß-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.

[Drug development from natural fermentation products: establishing a manufacturing process which maximizes the potential of microorganisms].

Natural fermentation products have long been studied as attractive targets for drug discovery due to their amazing diverse, complex chemical structures and biological activities. As such, a number of revolutionary drugs developed from natural fermentation products have contributed to global human health. To commercialize a drug derived from natural fermentation products, an effective chemical entity must be identified and thoroughly researched, and an effective manufacturing process to prepare a commercial supply must be developed. To construct such a manufacturing process for tacrolimus and micafungin, the following studies were conducted: first, we focused on controlling the production of the tacrolimus-related compound FR900525, a fermentation by-product of tacrolimus which was critical for quality assurance of the drug substance. FR900525 production was reduced by using a mutant strain which produced more pipecolic acid, the biosynthesis material of tacrolimus, than the original strain. Then, to optimize the fermentation process of FR901379, an intermediate of micafungin, a fed-batch culture was adopted to increase FR901379 productivity. Additionally, FULLZONE(TM) impeller was installed into the scaled-up fermenter, reducing the agitation-induced damage to the mycelium. As a result, the mycelial form changed from filamentous to pellet-shaped, and the air uptake rate during fermentation was drastically improved. Finally, we conducted screening for FR901379 acylase-producing microorganisms, as FR901379 acylase is necessary to manufacture micafungin. We were able to easily discover FR901379 acylase-producing microorganisms in soil samples using our novel, convenient screening method, which involves comparing the difference in antibiotic activity between FR901379 and its deacylated product.

Scale-up fermentation of echinocandin type antibiotic FR901379.

Industrial-scale production of FR901379 (WF11899A), which is a novel echinocandin type of lipopeptide antibiotic produced by mutant strain M-7 from Coleophoma empetri F-11899 (FERM BP-2635), was demonstrated. In order to achieve high-level production in fermentor culture, the medium previously developed was modified, in which three types of organic nitrogen were replaced by ammonium sulfate and corn steep liquor. To eliminate increase in viscosity, carbon source was intermittently fed. The viscosity was reduced from 20,000 cP to less than 10,000 cP. The FULLZONE impeller was introduced in the fermentor culture for sufficient mixing. Mixing time was quite improved and high reproducibility was achieved. Surprisingly, the viscosity of the broth was reduced to 1000 cP in a 4 m(3) scale fermentor. When k(L)a was selected as an index for scale-up and industrial scale production using a 15 m(3) fermentor with the FULLZONE impeller was conducted, FR901379 production was successfully obtained at more than 50 U/mL, almost the same level as with the 0.03 m(3) and 4 m(3) fermentors. In addition, superior reproducibility was obtained, and 500-fold scale-up was successfully achieved.

Screening and characterization of microorganisms with FR901379 acylase activity.

A simple and rapid screen was performed for microorganisms producing cyclic lipopeptide acylase, an enzyme that deacylates the acyl side chain of FR901379 to yield a cyclic peptide, FR179642, which is an important intermediate in producing micafungin. Among approximately 3800 newly isolated strains from soil samples, 5 microorganisms expressing high acylase activity were selected and classified, 3 as Streptomyces spp. and 2 as filamentous fungi. This is the first time that these strains have been identified as cyclic lipopeptide acylase producers. Both fungi and streptomycetes showed differing optimum pH and temperature profiles for acylase activity.

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.

Improvement of arterial stiffness by the antioxidant and anti-inflammatory effects of short-term statin therapy in patients with hypercholesterolemia.

The preventive effect of statins on coronary events is not only associated with the cholesterol-lowering effect of these drugs, but also various direct effects on the vascular wall, which include improvement of endothelial function, antioxidant activity, and anti-inflammatory activity. We investigated whether short-term statin therapy could improve arterial stiffness and assessed its mechanism of action in patients with hypercholesterolemia. We assessed arterial stiffness in 10 patients (mean age: 62.9 +/- 9.0 years) with hypercholesterolemia (total cholesterol > or =220 mg/dl). The patients were treated with cerivastatin (0.15 mg/day) for 4 weeks. Before and after 4 weeks of treatment, we determined arterial stiffness from brachial-ankle pulse wave velocity and the ankle-brachial blood pressure index (ABI) using a FORM apparatus (Colin, Komaki, Japan). We also measured the blood levels of high-sensitivity C-reactive protein (hsCRP) and malondialdehyde low-density lipoprotein (MDA-LDL) as markers of inflammation and oxidation, respectively. After statin therapy, both the right and left abPWV were significantly decreased from 1544.6 +/- 157.1 to 1349.0 +/- 223.9 cm/s and from 1592.1 +/- 164.8 to 1424.8 +/- 245.2 cm/s, respectively (P < 0.05). However, the ABI was unchanged after 4 weeks of cerivastatin therapy. MDA-LDL decreased significantly (from 161.2 +/- 42.4 to 119.4 +/- 33.5 U/l, P < 0.05) and hsCRP also decreased. Total cholesterol and LDL-cholesterol decreased, while triglycerides and high-density lipoprotein-cholesterol were unchanged. Blood pressure was not significantly altered from the baseline value by statin therapy. These results suggest that the preventive effect of statins on coronary events is partly associated with the various actions of these drugs on the vascular wall, and that statins are not only cholesterol-lowering agents but also antiatherosclerotic agents.