Eurycomanone from Eurycoma longifolia Jack upregulates neurotrophin-3 gene expression in retinal Müller cells in vitro.

Photoreceptor degeneration decreases light sensitivity and leads to vision loss and various retinal diseases. Neurotrophin-3, originating from Müller glial cells in the retina, plays a key role in protecting photoreceptors from damage induced by light or hypoxia. This neuroprotective approach is important because there are no established methods to regenerate lost photoreceptors. Dietary supplements are one of the useful methods for improving eye health. Eurycoma longifolia (E. longifolia) Jack, which is native to the tropical forest of Malaysia and other Southeast Asian countries, exhibits several medicinal properties. In the present study, we demonstrated that the water extract of E. longifolia roots enhanced neurotrophin-3 gene expression in primary rat Müller cells. Using a stepwise bioassay-guided fractionation and purification of E. longifolia root extracts, we isolated the active compound underlying neurotrophin-3 gene-enhancing activities. Mass spectrometry and nuclear magnetic resonance spectral data identified the compound as eurycomanone. This study provides evidence for the efficacy of E. longifolia and eurycomanone in enhancing neurotrophin-3 expression in Müller cells in vitro. Although the biological significance of this effect and its underlying mechanism remain to be elucidated, this study suggests that E. longifolia may be promising for improving eye health and must be further investigated.

Wood cauliflower mushroom (Sparassis crispa) suppresses the body weight and visceral fat increased by ovariectomy in mice.

Sparassis crispa, an edible mushroom, has been reported to show many kinds of physiological functions. The present paper focused on reducing body weight, subcutaneous fat, and visceral fat gain in ovariectomized (OVX) mice. Using the fruiting body powder of the indoor cultivation S. crispa (IT S. crispa: ITSc), one week after the OVX, ITSc was administered to two OVX groups by per os (p.o). In the sham group, 10 mL/kg water and 10 mL/kg saline were administered by p.o. and subcutaneous adm, respectively. OVX groups were divided into four groups. These treatments were performed on animals 6 days a week for 8 weeks. Subcutaneous and visceral fat measurements were performed under inhalation anesthesia with isoflurane using a Latheta LCT-200 X-ray CT system. The biochemical markers and the mRNA expression levels of the PPARγ, adiponectin, TNF-α, PPARα, and leptin were measured. Significant increases in body weight, fat ratio, and glucose levels were detected in OVX mice compared to sham mice. These increases were significantly blocked by ITSc, but not estradiol. Furthermore, ITSc treatment significantly increased adiponectin and leptin levels in adipose tissue. These results suggest that ITSc improves lipid abnormalities due to the less activity of women's ovary function, excluding estrogen functions.

2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Human Recombinant Lactoferrin Promotes Differentiation and Calcification on MC3T3-E1 Cells.

Lactoferrin (LF), known to be present in mammalian milk, has been reported to promote the proliferation of osteoblasts and suppress bone resorption by affecting osteoclasts. However, the mechanisms underlying the effects of human sources LF on osteoblast differentiation have not yet been elucidated, and almost studies have used LF from bovine sources. The presented study aimed to characterize the molecular mechanisms of bovine lactoferrin (IF-I) and human recombinant lactoferrin (LF-II) on MC3T3-E1 pre-osteoblast cells. MC3T3-E1 cells were treated with LF, ascorbic acid, and ß-glycerophosphate (ß-GP). Cell proliferation was analyzed using the MTT assay. Alkaline phosphatase activation and osteopontin expression levels were evaluated via cell staining and immunocytochemistry. The differentiation markers were examined using quantitative real-time PCR. The cell viability assay showed the treatment of 100 µg/mL LF significantly increased; however, it was suppressed by the simultaneous treatment of ascorbic acid and ß-GP. Alizarin red staining showed that the 100 µg/mL treatment of LF enhanced calcification. Quantitative real-time PCR showed a significant increase in osterix expression. The results suggest that treatment with both LFs enhanced MC3T3-E1 cell differentiation and promoted calcification. The mechanisms of calcification suggest that LFs are affected by an increase in osterix and osteocalcin mRNA levels.

Zinc-containing Mohs' paste affects blood flow and angiogenesis suppression.

PURPOSE: Mohs' paste, which is composed of zinc chloride and zinc oxide starch, is used for hemostasis of superficial malignancy in the clinical setting. We investigated the concentration of intramuscular zinc in mice after Mohs' paste application and evaluated its relationship with angiogenesis from the perspective of blood flow levels within 24 h. METHODS: Male C57BL/6JJmsSlc mice were administered single dose of Mohs' paste at 25%, 50%, and 75% after unilateral hind limb surgery, and glycerin, a viscosity modifier, was administered to the control group (0%). Hind limb blood flow levels were measured with a laser Doppler perfusion imaging system (n = 6). The amounts of intramuscular zinc and vascular endothelial growth factor-A (VEGF-A) expression were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) and western blotting, respectively (n = 5 or 3). RESULTS: Blood flow levels were significantly decreased in the 50% group after 8 h, and significantly decreased in the 25% and 50% groups after 24 h. Intramuscular zinc was significantly increased in the 50% and 75% groups after 8 h. Western blotting showed that VEGF-A levels were significantly increased in the 25% and 50% groups after 8 h. Based on analytical experiments and biological investigation, we predicated the pharmacological effect of Mohs' paste and found over 50% of it is critical in the blood flow and angiogenesis suppression after more than 8 h of its application. CONCLUSIONS: The results suggest that the mechanism of blood flow suppression is independent of VEGF-A levels and might suppress future angiogenesis. Our findings support that of previous studies, in which Mohs' paste was expected to induce hemostasis and suppress angiogenesis. It is an excellent ointment that facilitates hemostasis by suppressing blood flow regardless of angiogenesis, and may be apt for situations where hemostasis is required in the clinical setting.

Therapeutic effects of sertraline on improvement of Ovariectomy-induced decreased spontaneous activity in mice.

We have already reported that ovariectomized (OVX) rats reduced the spontaneous activity during the dark period due to the decease of serotonin release in the amygdala. In this study, we examined the potential of sertraline, a selective serotonin reuptake inhibitor, on the recovery of less spontaneous activity seen in mice with OVX-induced despair-like behaviors. Female 9-week old ICR mice were underwent either OVX or sham surgery. Sertraline (10 mg/kg/day, s.c.) or saline were started to administer to each group for 8 weeks (6 times/week) from the 8th week after OVX. Each spontaneous activity of mouse was evaluated during the dark period (19:00-07:00) using an infrared sensor. Moreover, mRNA expression levels of tryptophan hydroxylase (TPH) and X-box binding protein 1 (XBP1) were measured in the hippocampus and prefrontal cortex using by a real-time PCR method. We found out that the OVX-induced despair-like behaviors were improved by the continuous administration of sertraline. After treatment of OVX, our real-time PCR data showed that sertraline significantly suppressed the upregulation of XBP1 expression levels in both hippocampus and prefrontal cortex, although this suppression of the downregulation of TPH expression levels was seen in only hippocampus. These results suggest that sertraline improves the decrease in spontaneous activity induced by OVX assessed by the hippocampus suppressing decreased serotonin synthesis in the serotonergic neuron.

Quality evaluation of Pinellia tuber by LC-TOF/MS targeted to ephedrine.

Pinellia tuber (PTE, , , , , , , , ) is derived from the tuber of Pinellia ternata Breitenbach (Araceae), which is a crude drug used in traditional Japanese Kampo medicine for the purpose of antiemesis and expectoration. Since the separation of ephedrine from PTE in 1978, it has been listed as a PTE component in textbooks and internet information. Therefore, there are harmful effects on appropriate use in clinical practice because PTE is dealt with as a crude drug for doping target, and traditional Japanese Kampo medicine containing PTE must be carefully administered to the elderly. However, since the 1978 published report, there has not been any report on the isolation of ephedrine from PTE and the interpretation of biosynthesis remains questionable. In the present study, we analyzed the PTE samples in market distribution products by LC-TOF/MS. From the analysis of the result of ephedrine's m/z 148.113 [M + H-H2O]+, PTE was not detected (n = 55, detection limit: 0.5 ppb). Additionally, the tuber of P. tripartite (PTR, ), the tuber of P. pedatisecta (PPE, ), Arisaema Tuber (ART, ), and the tuber of Typhonium flagelliforme (TFI, ) that have a similar description to PTE were also not detected. Moreover, the genetic analysis of experimental samples showed that PTE is derived from P. ternata. Furthermore, our attempt to isolate ephedrine from PTE based on the past literature was unsuccessful. These results suggest that PTE in market distribution products may not contain ephedrine as a component.

Lactoferrin Suppresses Decreased Locomotor Activities by Improving Dopamine and Serotonin Release in the Amygdala of Ovariectomized Rats.

BACKGROUND: Decreases in female hormones not only affect bone metabolism and decrease bone mass, but also affect the central nervous system, causing brain disorders such as depression and dementia. Administration of estradiol by hormone replacement therapy can improve dementia, while reduced estradiol in ovariectomized (OVX) model rats can reduce both bone density and locomotor activity. The antidepressant fluvoxamine, which is widely used in clinical practice, can improve this effect on locomotor reduction. Similarly, lactoferrin (LF) can reportedly improve inhibitory locomotion due to stress. OBJECTIVE: In this study, we examined the effect of LF on neurite outgrowth in vitro and in vivo using PC12 cells and rats, respectively. METHODS: We performed an in vivo study in which 8-week-old female OVX rats were administered LF five days a week for 6 weeks from the day after surgery. After administration was completed, spontaneous locomotor activity in the dark period, immobility time in a forced swim test, and release amount of dopamine and serotonin in the brain were measured. RESULTS: LF was found to have a neurite outgrowth function in PC12 cells. Moreover, LF was found to improve OVX-induced decreases in locomotor activity and increases in immobility time in the forced swim test. Furthermore, the administration of LF elicited significant recovery of decreased dopamine and serotonin release in the brains of OVX group rats. CONCLUSION: These results strongly suggest that LF improved OVX-induced decreases in momentum during the dark period and, moreover, that release of dopamine and serotonin in the brain was involved in this effect.

Effects of the linagliptin, dipeptidyl peptidase-4 inhibitor, on bone fragility induced by type 2 diabetes mellitus in obese mice.

Recently, it has been suggested that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), which play important roles in the homeostasis of glucose metabolism, could be involved in the regulation of bone metabolism. Inhibitors of dipeptidyl peptidase 4 (DPP-4), an enzyme that degrades GIP and GLP-1, are widely used clinically as a therapeutic agent for diabetes. However, the effects of DPP-4 inhibitors on bone metabolism remain unclear. In this study, we investigated the effects of linagliptin, a DPP-4 inhibitor, on bone fragility induced by type 2 diabetes mellitus (T2DM). Non-diabetic mice were used as controls, and T2DM mice were administered linagliptin orally on a daily basis for 12 weeks. In T2DM mice, decreased bone mineral density was observed in the lower limb bones along with low serum osteocalcin levels and high serum tartrate-resistant acid phosphatase-5b (TRAP) levels. In contrast, the decreased serum osteocalcin levels and increased serum TRAP levels observed in T2DM mice were significantly suppressed after the administration of linagliptin 30 mg/kg. Bone histomorphometric analysis revealed a reduced osteoid volume and osteoblast surface with an increase in the eroded surface and number of osteoclasts in T2DM mice. This decreased bone formation and increased bone resorption observed in the T2DM mice were suppressed and trabecular bone volume increased following the administration of 30 mg/kg linagliptin. Collectively, these findings suggest that linagliptin may improve the microstructure of trabecular bone by inhibiting both a decrease in bone formation and an increase in bone resorption induced by T2DM.

Antiproliferative and Antimigration Activities of Beauvericin Isolated from Isaria sp. on Pancreatic Cancer Cells.

This study describes the antiproliferative and antimigration effects of beauvericin from a culture broth of Isaria sp. in human pancreatic cancer cells (PANC-1). Activity-guided fractionation of the EtOAc extract of cultured broth of Isaria sp. RD055140 afforded beauvericin (1), a new isariotin derivative, 7-O-methylisariotin C (2), together with the known isariotin analogs, TK-57-164A (3) and B (4). As a result of the measurement of the cell viability, 1 inhibited cell growth (IC50 = 4.8 µM) of PANC-1 cells. Furthermore, 1 was found to inhibit the migration activity of PANC-1 cells by upregulating the expression of the E-cadherin gene and reducing N-cadherin and Snail genes in a dose-dependent manner (0.1-1 µM). These activities of 1 had lower concentrations than the cytotoxic activity. These findings suggest that 1 can be used as an anticancer agent against human pancreatic carcinoma.

Effects of the combined administration of risedronate and menatetrenone on bone loss induced by tacrolimus in rats.

Tacrolimus, a calcineurin inhibitor, affects bone metabolism and increases the risk of fracture due to marked bone loss. Bisphosphonates increase the bone mineral density (BMD) in osteoporosis patients. Menatetrenone has less positive effects on BMD but reduces the risk of fracture by improving bone quality. In this study, we investigated the effectiveness of the combined administration of risedronate and menatetrenone against bone loss induced by tacrolimus. Wistar rats were divided into four groups: [1] control, [2] tacrolimus at 1.5 mg/kg, [3] tacrolimus + risedronate at 1.0 mg/kg, and [4] tacrolimus + risedronate + menatetrenone at 20 mg/kg. After the drugs were administered for 4 weeks, bone histomorphometric analysis was performed and bone strength was evaluated using a three point bending method. BMD was measured using quantitative computed tomography. Tacrolimus significantly reduced the BMD and strength properties of the lower limb bones. These tacrolimusinduced decreases were suppressed by risedronate treatment. The combined administration of risedronate and menatetrenone more significantly improved bone strength properties than risedronate alone. Bone histomorphometric analysis revealed a significant increase in bone resorption with tacrolimus. Risedronate alone significantly suppressed the tacrolimus-induced increase in bone resorption but simultaneously reduced bone formation. On the other hand, the combined administration of risedronate and menatetrenone suppressed the tacrolimus-induced increase in bone resorption, in addition to the significant risedronate-induced decrease in bone formation. This study suggests that the combined administration of risedronate and menatetrenone improves bone strength in tacrolimus-treated rats by preventing and ameliorating the risedronate-induced suppression of bone formation.

Effects of Coriandrum sativum Seed Extract on Aging-Induced Memory Impairment in Samp8 Mice.

The purpose of this study was to investigate whether or not Coriandrum sativum seed extract (CSSE) can ameliorate memory impairment in senescence-accelerated mouse-prone 8 (SAMP8) mice. Sixteen 10-week-old male SAMP8 mice were divided into two groups, which were orally administrated water (SAMP8(-)) or CSSE (200 mg/kg/day; SAMP8(+)). Eight 10-week-old male Institute of Cancer Research (ICR) mice were used as a normal control group and were also orally administrated water. The mean escape time in the Barnes maze test of SAMP8(-) mice was significantly longer than that of ICR mice. However, SAMP8(+) mice showed a shorter mean escape time compared to that of SAMP8(-) mice. Neurofilament messenger (m)RNA levels significantly decreased in the frontal lobe of SAMP8(-) mice when compared with ICR mice, but significantly increased in SAMP8(+) mice relative to SAMP8(-) mice. In addition, mRNA levels of inducible nitric oxide synthase (iNOS) and neuronal (n)NOS significantly increased in the frontal lobe of SAMP8(-) mice, but only the mRNA level of nNOS significantly decreased in SAMP8(+) mice. These results indicated that continuous oral administration of CSSE for 12 weeks could ameliorate aging-induced memory declines in the senescence-accelerated SAMP8 mouse model.

Bile Acid 7α-Dehydroxylating Gut Bacteria Secrete Antibiotics that Inhibit Clostridium difficile: Role of Secondary Bile Acids.

Clostridium scindens biotransforms primary bile acids into secondary bile acids, and is correlated with inhibition of Clostridium difficile growth in vivo. The aim of the current study was to determine how C. scindens regulates C. difficile growth in vitro and if these interactions might relate to the regulation of gut microbiome structure in vivo. The bile acid 7α-dehydroxylating gut bacteria, C. scindens and C. sordellii, were found to secrete the tryptophan-derived antibiotics, 1-acetyl-ß-carboline and turbomycin A, respectively. Both antibiotics inhibited growth of C. difficile and other gut bacteria. The secondary bile acids, deoxycholic acid and lithocholic acid, but not cholic acid, enhanced the inhibitory activity of these antibiotics. These antibiotics appear to inhibit cell division of C. difficile. The results help explain how endogenously synthesized antibiotics and secondary bile acids may regulate C. difficile growth and the structure of the gut microbiome in health and disease.

Absorption, disposition and metabolic pathway of amiselimod (MT-1303) in healthy volunteers in a mass balance study.

The absorption, metabolism and excretion of MT-1303 were investigated in healthy male subjects after a single oral dose of 0.4 mg [14C]-MT-1303 (ClinicalTrials.gov NCT02293967). The MT-1303 concentration in the plasma reached a maximum at 12 h after administration. Thereafter, the concentration declined with a half-life of 451 h. At the final assessment on Day 57, 91.16% of the administered radioactivity was excreted, and the cumulative excretion in the urine and faeces was 35.32% and 55.84%, respectively. The most abundant metabolite in plasma was MT-1303-P, which accounted for 42.6% of the area under the plasma concentration-time curve (AUC) of the total radioactivity. The major component excreted in urine was Human Urine (HU)4 (3066434), accounting for 28.1% of radioactivity in the sample (4.05% of the dose), whereas MT-1303 was a major component in the faeces, accounting for 89.8% of radioactivity in the sample (25.49% of the dose) up to 240 h after administration. This study indicates that multiple metabolic pathways are involved in the elimination of MT-1303 from the human body and the excretion of MT-1303 and MT-1303-P via the kidney is low. Therefore, MT-1303 is unlikely to cause conspicuous drug interactions or alter pharmacokinetics in patients with renal impairment.

Effects of the calcineurin inhibitors cyclosporine and tacrolimus on bone metabolism in rats.

Immunosuppressive therapy is considered as one of the factors inducing to the onset of osteoporosis after organ transplantation. Chronic immunosuppressive therapy after transplantation is required for organ transplant patients, and it is important to prevent the occurrence of osteoporotic fractures to maintain the quality of life in patients. In this study, we examined the effects of cyclosporine and tacrolimus on bone metabolism in rats. Five-week-old male Wistar rats were treated orally with 15 mg/kg cyclosporine or 1.5 mg/kg tacrolimus daily for 4 weeks. Each of cyclosporine and tacrolimus significantly reduced the bone strength of the femoral mid-diaphysis and bone mineral density of the tibia and femur. Bone histomorphometry showed that the administration of both drugs resulted in a decrease in bone volume, number and thickness of trabeculae, and an increase in trabecular separation. Bone formation parameters such as osteoid volume, osteoblast surface, mineralizing surface, mineral apposition rate, and bone formation rate significantly increased in the cyclosporine-treated group. Bone resorption parameters such as eroded surface, osteoclast surface, and osteoclast number significantly increased in both the cyclosporine- and the tacrolimus- treated groups. These results showed that cyclosporine increases both bone formation and bone resorption, leading to a high-turnover bone loss, and that tacrolimus increases bone resorption without affecting bone formation, leading to bone loss.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Triterpenoid glycosides from Ladenbergia hexandra Klotzsch.

From the bark of Ladenbergia hexandra Klotzsch, ten triterpenoid glycosides were isolated along with five known compounds, and their structures were determined based on extensive NMR and mass spectroscopic, GC and HPLC analyses. Some triterpenoid glycosides contained 6-deoxy-D-allose or D-allose as a sugar moiety. The absolute stereochemical assignment of the sugars was determined by comparison with synthetic samples, as well as by GC and HPLC analysis.

A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship.

To explore the structure-activity relationships on the inhibitory activity of flavanonols against nitric oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common 3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to investigate the structure-activity relationship. We also succeeded in isolating stereoisomers from 16 of the flavanonols using chiral column chromatography. The inhibitory effects of these compounds on NO production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were activated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in (2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker. We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of NO production by flavanonols is stereospecific and cannot simply be explained by their radical scavenging activity. We propose the possible existence of a 'target' molecule for flavanonols which is involved in the production and/or regulation of NO in RAW 264.7 cells.

Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.