Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Emulsifiers and thickeners on extrusion-cooked instant rice product.

Extrusion-cooked instant rice was prepared by optimizing the formulation with emulsifiers, glycerol monostearate (GMS), soybean lecithin (LC), and sodiumstearoyl lactylate (SSL), and thickeners, gum Arabic (GA), sodium alginate (SA), and sticky rice (SR). The emulsifiers addition caused increase of degree of gelatinization (DG), and decrease of water soluble carbohydrate (WSC), α-amylase sensitivity, water soluble index (WAI) and adhesive for extrudates, while the thickeners addition increased extrudates DG, bulk density (BD), WSC, α-amylase sensitivity, WAI, hydration rate (HR) and adhesiveness. Based on the data generated by a single additive at various levels, optimum formulation was obtained employing orthogonal matrix system with combination of the selected additives for extrusion cooking. Extrudates were evaluated for optimum hydration time followed by drying to prepare the finished product. Texture profile analysis and sensory evaluation indicate that quality of the finished product is equivalent to that of the round shaped rice and superior to a commercial instant rice product. This study also demonstrates possibility of value-added and versatile instant rice product development using broken rice.

Licochalcone E has an antidiabetic effect.

Licochalcone E (lico E) is a retrochalcone isolated from the root of Glycyrrhiza inflata. Retrochalcone compounds evidence a variety of pharmacological profiles, including anticancer, antiparasitic, antibacterial, antioxidative and superoxide-scavenging properties. In this study, we evaluated the biological effects of lico E on adipocyte differentiation in vitro and obesity-related diabetes in vivo. We employed 3T3-L1 preadipocyte and C3H10T1/2 stem cells for in vitro adipocyte differentiation study and diet-induced diabetic mice for in vivo study. The presence of lico E during adipogenesis induced adipocyte differentiation to a significant degree, particularly at the early induction stage. Licochalcone E evidenced weak, but significant, peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding activity. Two weeks of lico E treatment lowered blood glucose levels and serum triglyceride levels in the diabetic mice. Additionally, treatment with lico E resulted in marked reductions in adipocyte size and increases in the mRNA expression levels of PPARγ in white adipose tissue (WAT). Licochalcone E was also shown to significantly stimulate Akt signaling in epididymal WAT. In conclusion, lico E increases the levels of PPARγ expression, at least in part, via the stimulation of Akt signals and functions as a PPARγ partial agonist, and this increased PPARγ expression enhances adipocyte differentiation and increases the population of small adipocytes, resulting in improvements in hyperglycemia and hyperlipidemia under diabetic conditions.

Nutritional evaluation of protein from Clanis bilineata (Lepidoptera), an edible insect.

BACKGROUND: This study was carried out to determine the nutritive quality of protein from Clanis bilineata (CB), an edible insect. RESULTS: The protein content of dried CB was 685.3 g kg(-1) and its essential amino acid content was 528.4 g kg(-1), both of which are comparable to those of milk and eggs. Three groups of rats were fed diets containing either casein or CB protein (CBP) as protein source or a protein-free diet for 10 days. The nutritional parameters measured for the group fed the diet containing CBP showed a positive nitrogen balance of 1.37, a net protein retention of 2.9 and a true digestibility of 95.8%, comparable to those measured for the group fed the casein diet. CONCLUSION: The results suggest that CBP may be a suitable alternative dietary protein source for humans.

Preparation of glucosamine by hydrolysis of chitosan with commercial α-amylase and glucoamylase.

OBJECTIVE: In order to overcome the defects of chemical hydrolysis approach to prepare glucosamine, an enzymatic hydrolysis method was developed. METHODS: Glucosamine was prepared by hydrolyzing chitosan, employing α-amylase initially, and subsequently, glucoamylase. RESULTS: The optimal hydrolyzing conditions were as follows: reaction time, 4 h; pH, 5.0; temperature, 50 °C; and, α-amylase, 80 U/g for the initial reaction. Subsequently, glucoamylase was added in the presence of α-amylase. The optimal reaction conditions were found to be: reaction time, 8 h; pH, 4.5; temperature, 55 °C; and, glucoamylase, 4000 U/g. The hydrolysates were subject to filtrating, concentrating to about 20% (w/w), precipitating with five volumes of ethanol, and drying at 60 °C for 2 h. The content and the yield of glucosamine in the dried precipitate were 91.3% (w/w) and 86.2% (w/w), respectively. CONCLUSIONS: The method developed in this study is a promising option in the preparation of glucosamine.

Estimation of pullulan by hydrolysis with pullulanase.

A novel method for the estimation of pullulan was developed in which pullulan was hydrolysed by pullulanase. The hydrolysed product was mainly maltotriose and was determined colorimetrically using 3,5-dimethylsalicylic acid. This gave good linearity with respect to the concentration of pullulan in the fermentation broth. The content of pullulan determined in this way was less than that determined by a coupled enzyme assay and was comparable to that determined by an HPLC method. The new method was specific for estimation of pullulan, demonstrated high accuracy, and could assay pullulan from up to 3.2 mg/ml.

Konjac glucomannan as a carrier material for time--temperature integrator.

Hardness, springiness and water retention of konjac glucomannan gel (g-KGM) as a novel carrier material for time-temperature integrator (TTI) in aseptic processing were determined and compared with those of sodium alginate gel (g-SA). Hardness of both g-KGM and g-SA increased with temperature: values of g-SA were significantly higher (p < 0.05) than those of g-KGM at all temperatures. No significant difference in springiness between g-KGM and g-SA from 40 °C to 90 °C and significant differences (p < 0.05) between 100 °C and 140 °C were found. Water retention property of g-KGM was lower than that of g-SA between 60 °C and 100 °C, but much higher between 100 °C and 140 °C. Heat transfer tests performed on g-KGM alone as well as on g-KGM as a carrier, embedded with TTI, α-amylase as an integrator, indicated that g-KGM was suitable for industrial ultrahigh temperature sterilization test.

Wogonin inhibits osteoclast formation induced by lipopolysaccharide.

To evaluate the inhibitory activity of wogonin against lipopolysaccharide (LPS)-induced bone resorption, we investigated the effect of wogonin on osteoclastogenesis induced by LPS. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures of mouse calvaria-derived osteoblasts and bone marrow-derived pre-osteoclasts. Wogonin also suppressed osteoclastogenesis in LPS-injected mouse calvaria. In osteoblasts, the upregulation of receptor activator of nuclear factor-kappaB (RANKL) expression and the downregulation of osteoprotegerin (OPG) expression by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPS-stimulated PGE(2) production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS-induced osteoclastogenesis through downregulation of RANKL and upregulation of OPG expression via blockage of PGE(2) production. Based on these results, wogonin has potential for use as a therapeutic agent in bacteria-induced bone resorption.

Changes in allele-specific association of histone modifications at the imprinting control regions during mouse preimplantation development.

Allele-specific association of histone modification is observed at the regulatory region of imprinted genes and has been suggested to work as an epigenetic marker for monoallelic gene expression, along with the allelic CpG methylation of DNA. Although the parent-origin-specific epigenetic status in imprinted genes is thought to be established during preimplantation development, little is known about the allelic specificity of histone modifications during this period because of the limited volume of material available for analysis. In this study, we first revealed the allelic enrichment of histone modifications and variant histones at the imprinting control regions (ICRs) of four-cell to blastocyst stage preimplantation embryos by using carrier chromatin immunoprecipitation and sequence polymorphism analysis of immunoprecipitated DNA. We found relative enrichment of histone H3 lysine 9 dimethylation at the imprinted alleles of ICRs and obtained the results suggesting that histone modifications at ICRs are established during a late preimplantation stage.

Mouse strain-dependent osteoclastogenesis in response to lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1alpha,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1alpha,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.

Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer.

In general, cloning undifferentiated preimplantation embryos (blastomeres) or embryonic stem cells is more efficient than cloning differentiated somatic cells. Therefore, there has been an assumption that tissue-specific stem cells might serve as efficient donors for nuclear transfer because of the undifferentiated state of their genome. Here, we show that this is not the case with adult hematopoietic stem cells (HSCs). Although we have demonstrated for the first time that mouse HSCs can be cloned to generate offspring, the birth rates (0-0.7%) were lowest among the clones tested (cumulus, immature Sertoli and fibroblast cells). Only 6% of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus clones; P < 5 x 10(-10)). Transcription and gene expression analyses of HSC clone embryos revealed that they initiated zygotic gene activation (ZGA) at the appropriate timing, but failed to activate five out of six important embryonic genes examined, including Hdac1 (encoding histone deacetylase 1), a key regulator of subsequent ZGA. These results suggest that the HSC genome has less plasticity than we imagined, at least in terms of reprogrammability in the ooplasm after nuclear transfer.

Regulation of histone acetylation during meiotic maturation in mouse oocytes.

Histone acetylation is an important epigenetic modification implicated in the regulation of chromatin structure and, subsequently, gene expression. Global histone deacetylation was reported in mouse oocytes during meiosis but not mitosis. The regulation of this meiosis-specific deacetylation has not been elucidated. Here, we demonstrate that p34(cdc2) kinase activity and protein synthesis are responsible for the activation of histone deacetylases and the inhibition of histone acetyltransferases (HATs), respectively, resulting in deacetylation of histone H4 at lysine-12 (H4K12) during mouse oocyte meiosis. Temporal changes in the acetylation state of H4K12 were examined immunocytochemically during meiotic maturation using an antibody specific for acetylated H4K12. H4K12 was deacetylated during the first meiosis, temporarily acetylated around the time of the first polar body (PB1) extrusion, and then deacetylated again during the second meiosis. Because these changes coincided with the known oscillation pattern of p34(cdc2) kinase activity, we investigated the involvement of the kinase in H4K12 deacetylation. Roscovitine, an inhibitor of cyclin-dependent kinase activity, prevented H4K12 deacetylation during both the first and second meiosis, suggesting that p34(cdc2) kinase activity is required for deacetylation during meiosis. In addition, cycloheximide, a protein synthesis inhibitor, also prevented deacetylation. After PB1 extrusion, at which time H4K12 had been deacetylated, H4K12 was re-acetylated in the condensed chromosomes by treatment with cycloheximide but not with roscovitine. These results demonstrate that HATs are present but inactivated by newly synthesized protein(s) that is (are) not involved in p34(cdc2) kinase activity. Our results suggest that p34(cdc2) kinase activity induces the deacetylation of H4K12 and that the deacetylated state is maintained by newly synthesized protein(s) that inhibits HAT activity during meiosis.

Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic marker of parental genome origin during early preimplantation development.

Significance of chin-brow vertical angle in correction of kyphotic deformity of ankylosing spondylitis patients.

STUDY DESIGN: A prospective study. OBJECTIVES: To assess the significance of chin-brow vertical angle in planning and evaluating the correction of kyphotic deformity with ankylosis of the cervical spine in ankylosing spondylitis patients. SUMMARY OF BACKGROUND DATA: Accurate assessment and measurement of spinal kyphotic deformity is required when planning treatment and assessing its results. METHODS: Thirty-four ankylosing spondylitis patients with cervical ankylosis who had undergone pedicle subtraction extension osteotomy for correction of kyphotic deformity were studied. Radiographic assessment for sagittal balance was performed by measuring thoracic kyphosis, lumbar lordosis, the distance between the vertical line on the anterosuperior point of T1 and that of S1, and sacral inclination. Chin-brow vertical angle was measured on the clinical photos of the patients. Clinical outcomes were assessed by a questionnaire. RESULTS: The preoperative and postoperative chin-brow vertical angles were 35.5 degrees and 1.8 degrees, respectively. Final follow-up radiographs showed an increase in lumbar lordosis from 5.5 degrees to 43.2 degrees (an increase of 37.7 degrees ), and thoracic kyphosis remained stable from 50.4 degrees to 50.2 degrees. Sagittal imbalance significantly improved from 101.5 mm to 12.7 mm. The decreased chin-brow vertical angle correlated negatively with the correction angle. The patients with a chin-brow vertical angle of less than -10 degrees had significantly low scores on horizontal gaze. CONCLUSIONS: Chin-brow vertical angle was an objective index for evaluating horizontal gaze. Based on the results of this study, measurement of chin-brow vertical angle is recommended for planning correction of kyphosis and accurate evaluation of treatment outcome.

Changes in histone acetylation during mouse oocyte meiosis.

We examined global changes in the acetylation of histones in mouse oocytes during meiosis. Immunocytochemistry with specific antibodies against various acetylated lysine residues on histones H3 and H4 showed that acetylation of all the lysines decreased to undetectable or negligible levels in the oocytes during meiosis, whereas most of these lysines were acetylated during mitosis in preimplantation embryos and somatic cells. When the somatic cell nuclei were transferred into enucleated oocytes, the acetylation of lysines decreased markedly. This type of deacetylation was inhibited by trichostatin A, a specific inhibitor of histone deacetylase (HDAC), thereby indicating that HDAC is able to deacetylate histones during meiosis but not during mitosis. Meiosis-specific deacetylation may be a consequence of the accessibility of HDAC1 to the chromosome, because HDAC1 colocalized with the chromosome during meiosis but not during mitosis. As histone acetylation is thought to play a role in propagating the gene expression pattern to the descendent generation during mitosis, and the gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the initiation of a new program by totipotent zygotes of the next generation, our results suggest that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones.

Analysis of the mechanism for chromatin remodeling in embryos reconstructed by somatic nuclear transfer.

The objective of the present study was to understand the molecular/biochemical nature of chromatin remodeling that occurs in the somatic nuclei transferred into oocytes. We produced the reconstructed mouse embryos by two different protocols of nuclear transfer. The nucleus of a cumulus cell was transferred into enucleated unfertilized oocytes (transferred before activation, TA protocol) or activated oocytes (activated before transfer, AT protocol). More than half (56.1%) of the embryos reconstructed using the TA protocol developed to the morula/blastocyst stage, whereas very few (1.0%) of the embryos reconstructed using the AT protocol reached the morula/blastocyst stage. These embryos were analyzed for the events associated with transcriptional regulation. Changes in transcriptional activity, nuclear accumulation of TATA box binding protein (TBP), and DNase I sensitivity were examined after nuclear transfer. In the embryos reconstructed by TA protocol, all of these events occurred in a manner similar to that in the control diploid parthenogenetic embryos. The transcriptional activity was silenced after nuclear transfer and resumed at the late 1-cell stage. TBP was displaced and subsequently accumulated at the early and the late 1-cell stage, respectively. DNase I sensitivity was increased and then decreased at the early and late 1-cell stage, respectively. In contrast, embryos reconstructed using the AT protocol did not show such changes in transcriptional activity, TBP accumulation, and DNase I sensitivity. These events would be necessary for differentiated nuclei to restore totipotency and are useful indices to evaluate successful chromatin remodeling.