Impact of Energy intake at One Week after Hospitalization on Prognosis for Older Adults with Pneumonia.

OBJECTIVES: This study objectives to investigate the influence of average energy intake at 1 week of hospitalization on prognosis for older adults with pneumonia. DESIGN: Retrospective observational cohort study. SETTING: The Japan Rehabilitation Nutrition Database comprise those with pneumonia in acute care hospitals. PARTICIPANTS: The study included 329 pneumonia patients (aged over 65 years) who entered into the Japan Rehabilitation Nutrition Database (JRND) from November 2015 to March 2018. MEASUREMENTS: Logistic regression analysis was performed to confirm the relationship of energy intake with the rate of mortality, discharge home, and pneumonia recurrence during hospitalization. Variables included in the multiple regression analysis model were age, sex, Mini Nutritional Assessment-Short Form score (MNA-SF) at hospitalization, A-DROP, Charlson comorbidity index (CCI), and presence or absence of rehabilitation. RESULTS: Of 315 patients with pneumonia (median age 85 years), 63.8% were men. 57.7% were assigned to the lack of energy intake (LEI) at 1 week after admission. Patients in the LEI group were older (p = 0.033), had higher A-DROP score (p < 0.001), and showed higher malnutrition rate in MNA-SF at hospitalization (p < 0.001) than those in the control group. Mortality, pneumonia recurrence (p = 0.001), median body mass index (p = 0.012), and low malnutrition in MNA-SF (p < 0.001) at discharge were significantly higher in the LEI group than in the control group. Logistic regression analysis showed that LEI was an independent risk factor for mortality (Odds ratio: 5.07, p = 0.002), discharge home (Odds ratio: 0.33, p = 0.007), and pneumonia recurrence (Odds ratio: 3.26, p = 0.007). CONCLUSIONS: LEI at 1 week after hospitalization in older adults with pneumonia was an independent risk factor for mortality, difficult at-home recovery, and pneumonia recurrence. These findings suggest the importance of adequate energy intake from the early days of hospitalization.

HLA-G1, but Not HLA-G3, Suppresses Human Monocyte/Macrophage-mediated Swine Endothelial Cell Lysis.

The inhibitory function of HLA-G1, a class Ib molecule, on monocyte/macrophage-mediated cytotoxicity was examined. The expression of inhibitory receptors that interact with HLA-G, immunoglobulin-like transcript 2 (ILT2), ILT4, and KIR2DL4 (CD158d) on in vitro-generated macrophages obtained from peripheral blood mononuclear cells and the phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells were examined by flow cytometry. cDNAs of HLA-G1, HLA-G3, HLA-E, and human ß2-microglobulin were prepared, transfected into pig endothelial cells (PECs), and macrophage- and the THP-1 cell-mediated PEC cytolysis was then assessed. In vitro-generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on PEC indicated a significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. HLA-G1 was clearly expressed on the cell surface of PEC, whereas the levels of HLA-G3 were much lower and remained in the intracellular space. On the other hand, the PMA-activated THP-1 cell was less expressed these inhibitory molecules than in vitro-generated macrophages. Therefore, the HLA-G1 on PECs showed a significant but relatively smaller suppression to THP-1 cell-mediated cytotoxicity compared to in vitro-generated macrophages. These results indicate that by generating HLA-G1, but not HLA-G3, transgenic pigs can protect porcine grafts from monocyte/macrophage-mediated cytotoxicity.

Supplemental Analysis for N-linked Sugars in Adult Pig Islets.

The pig pancreas is considered to be one of the most suitable sources of islets for clinical xenotransplantation. However, after producing α1-3galactosyltransferase knockout pigs, most of the organs of these pigs showed less antigenicity to the human body. Wild-type adult pig islets (APIs) that originally produced negligible levels of α-Gal, different from neonatal porcine islet-like cell clusters, showed a clear antigenicity to human serum. Concerning the so-called non-Gal epitopes, many studies related to glycoproteins and glycolipids are ongoing in efforts to identify them. However, our knowledge of non-Gal glycoantigens remains incomplete. In our previous study, N-glycans were isolated from APIs, and the structures of 28 of the N-glycans were detected. In this study, to identify additional structures, further analyses were performed by liquid chromatography-mass spectrometry (LC-MS). N-glycans were isolated from APIs by the method described by O'Neil et al with minor modifications and LC-MS-based structural analyses were then performed. The detected N-glycan peaks in the LC-MS spectra were selected using the FLexAnalysis software program and the structures of the glycans were predicted using the GlyocoMod Tool. The API preparation contained 11 peaks and 16 structures were then nominated as containing N-linked sugars. Among them, 5 sulfated glycans were estimated, confirming the existence of sulfate structures in N-glycans in API. In addition, these data may supplement several N-glycan structures that contain two deoxyhexose units, such as fucose, to our previous report. The data herein will be helpful for future studies of antigenicity associated with API.

Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System.

BACKGROUND: We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). METHODS: Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. RESULTS: Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. CONCLUSIONS: Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.

Effect of glycogen synthase kinase 3 ß-mediated presenilin 1 phosphorylation on amyloid ß production is negatively regulated by insulin receptor cleavage.

Presenilin 1 (PS1), a causative molecule of familial Alzheimer's disease (AD), is known to be an unprimed substrate of glycogen synthase kinase 3 ß (GSK3ß) [Twomey and McCarthy (2006) FEBS Lett 580:4015-4020] and is phosphorylated at serine 353, 357 residues in its cytoplasmic loop region [Kirschenbaum et al. (2001) J Biol Chem 276:7366-7375]. In this report, we investigated the effect of PS1 phosphorylation on AD pathophysiology and obtained two important results--PS1 phosphorylation increased amyloid ß (Aß) 42/40 ratio, and PS1 phosphorylation was enhanced in the human AD brains. Interestingly, we demonstrated that PS1 phosphorylation promoted insulin receptor (IR) cleavage and the IR intracellular domain (IR ICD) generated by γ-secretase led to a marked transactivation of Akt (PKB), which down-regulated GSK3ß activity. Thus, the cleavage of IR by γ-secretase can inhibit PS1 phosphorylation in the long run. Taken together, our findings indicate that PS1 phosphorylation at serine 353, 357 residues can play a pivotal role in the pathology of AD and that the dysregulation of this mechanism may be causally associated with its pathology.

Hypertriglyceridemia with increased plasma insulin concentrations in cats.

Metabolite, insulin and adiponectin concentrations and LDH, AST and ALT activities were measured in plasma of 142 client-owned cats (1-13years old, 16 breeds) to set up a new criterion of hypertriglyceridemia (hyper-TG) with increased plasma insulin concentrations for early diagnosis of lipid metabolism abnormality including obesity. 25 cats with over 165mg/dl of plasma triglyceride (TG) concentrations were decided as hyper-TG with increased plasma insulin concentrations, and prevalence of hyper-TG was 16.7% in young (1-6years old) and 18.3% in old (>7years old) cats examined. In the hyper-TG cats, their plasma TG concentrations increased to 6.6-7.4-fold of the values of control cats with 35-50mg/dl of plasma TG and their plasma cholesterol, FFA and insulin concentrations and LDH and ALT activities increased significantly, whereas their plasma adiponectin concentrations decreased significantly compared to those in the control cats. Hyper-TG cats with significantly increased body weights and plasma insulin and decreased plasma adiponectin seemed to be in early stage of obesity accompanying increased plasma insulin concentrations. Increased TG, insulin, LDH and ALT and decreased adiponectin values in plasma seemed to be key factors for diagnosis of lipid metabolism abnormality at early stage in cats.

Comparison of plasma metabolite concentrations and enzyme activities in beef cattle raised by different feeding systems in Korea, Japan and New Zealand.

Concentrations of metabolites and immunoreactive insulin (IRI) and activities of enzymes related to energy metabolism were measured in plasma of Korean and Japanese beef cattle, which were raised by the indoor feeding system programmed to feed larger amount of roughage in their growing periods and larger amount of concentrate diet in their finishing periods (Japanese feeding system), and grazing New Zealand beef cattle. By the Japanese beef grading system, Korean and Japanese beef cattle showed high beef quality score, average grade 3.3 and 3.6, respectively. The plasma free fatty acid and lactate concentrations and lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and aspartate aminotransferase (AST) activities in Korean beef cattle were significantly higher than those in Japanese beef cattle. The plasma lactate concentration in Korean beef cattle was 8.40 mmol/l, which was similar to the values observed in lactic acidosis. The higher activities of plasma LDH, MDH and AST may indicate slight liver damage by slightly acidotic conditions in Korean beef cattle. New Zealand beef cattle fed on pasture which they harvest by grazing showed significantly lower plasma glucose, cholesterol, lactate and IRI concentrations and enzyme activities than those in Korean and Japanese beef cattle fed on larger amount of concentrate diets. Plasma metabolite concentrations and energy metabolism-related enzyme activities may be good indicators for evaluating metabolic conditions of beef cattle raised by different feeding systems.

Hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells.

BACKGROUND AND PURPOSE: A traditional Japanese herbal medicine, hochu-ekki-to, has been used for the symptomatic treatment of the common cold and to reduce the frequency of colds in patients with chronic obstructive pulmonary disease. However, the inhibitory effects of hochu-ekki-to on infection by rhinovirus (RV), the major cause of common colds, have not been studied. EXPERIMENTAL APPROACH: Human tracheal epithelial cells in culture were infected with a major group rhinovirus-RV14. Virus output and viral RNA were measured along with interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha), mRNA for intercellular adhesion molecule (ICAM)-1 and acidic endosomes in cells. KEY RESULTS: RV14 infection increased virus titers, the content of cytokines in supernatants and RV14 RNA in the cells. Hochu-ekki-to decreased virus output, RV14 RNA in the cells, susceptibility to RV infection and supernatant cytokine concentrations after RV14 infection. Hochu-ekki-to reduced mRNA for ICAM-1, the receptor for RV14, the concentration of the soluble form of ICAM-1 and the number and fluorescence intensity of acidic endosomes in the cells, from which RV RNA enters into the cytoplasm, at RV14 infection. Glycyrrhizin, one of the chemical constituents of hochu-ekki-to, reduced supernatant virus titers dose-dependently. CONCLUSION AND IMPLICATIONS: Hochu-ekki-to inhibited RV14 infection by decreasing ICAM-1 and by blocking entry of viral RNA into the cytoplasm from the endosomes, in airway epithelial cells. Glycyrrhizin may be partly responsible for inhibition of RV infection by hochu-ekki-to. Hochu-ekki-to could modulate airway inflammation by reducing production of cytokines in RV infections.

Association of susceptibility to the development of pneumonia in the older Japanese population with haem oxygenase-1 gene promoter polymorphism.

BACKGROUND: Oxidative stresses including cigarette smoking are implicated in the pathogenesis of cerebrovascular diseases, which are associated with pneumonia because of frequent aspiration. Haem oxygenase-1 (HO-1) acts in cytoprotection against oxidants, provides anti-inflammatory effects, and inhibits atherogenesis. A (GT)(n) dinucleotide repeat in the human HO-1 promoter modulates HO-1 gene expression and shows length polymorphism, which is grouped into three classes: class S (<27 repeats), class M (> or = 27, <33 repeats), and class L (> or = 33 repeats) alleles. OBJECTIVE: To investigate the correlation between the HO-1 gene polymorphism and development of pneumonia in elderly Japanese. METHODS: The length of the (GT)n repeats was analysed in 200 elderly patients with pneumonia and 200 control subjects. The association of the HO-1 gene polymorphism with risk of pneumonia was estimated by logistic regression. RESULTS: The proportion of allele frequencies in class L, and the proportion of genotypic frequencies in the L-allele carriers (L/L, L/M, and L/S), was significantly higher in patients with pneumonia than in controls (20% v 10% in class L, and 34% v 18% in L-allele carriers). After adjustment for potentially confounding factors, both cerebrovascular disorders and HO-1 gene L-allele carriers were significant and independent risk factors for pneumonia. The adjusted odds ratio for L-allele carriers v non-L-allele carrier was 2.1 (95% confidence interval, 1.2 to 3.6). CONCLUSIONS: The large size of a (GT)n repeat in the HO-1 gene promoter may be associated with susceptibility to pneumonia in the older Japanese population.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells.

The aim of the study was to examine the effects of a mucolytic drug, carbocisteine, on rhinovirus (RV) infection in the airways. Human tracheal epithelial cells were infected with a major-group RV, RV14. RV14 infection increased virus titres and the cytokine content of supernatants. Carbocisteine reduced supernatant virus titres, the amount of RV14 RNA in cells, cell susceptibility to RV infection and supernatant cytokine concentrations, including interleukin (IL)-6 and IL-8, after RV14 infection. Carbocisteine reduced the expression of mRNA encoding intercellular adhesion molecule (ICAM)-1, the receptor for the major group of RVs. It also reduced the supernatant concentration of a soluble form of ICAM-1, the number and fluorescence intensity of acidic endosomes in the cells before RV infection, and nuclear factor-kappaB activation by RV14. Carbocisteine also reduced the supernatant virus titres of the minor group RV, RV2, although carbocisteine did not reduce the expression of mRNA encoding a low density lipoprotein receptor, the receptor for RV2. These results suggest that carbocisteine inhibits rhinovirus 2 infection by blocking rhinovirus RNA entry into the endosomes, and inhibits rhinovirus 14 infection by the same mechanism as well as by reducing intercellular adhesion molecule-1 levels. Carbocisteine may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

Gastric mucosal hyperplasia via upregulation of gastrin induced by persistent activation of gastric innate immunity in major histocompatibility complex class II deficient mice.

BACKGROUND AND AIMS: Major histocompatibility complex class II deficient (Aalpha0/0) mice have decreased CD4+ T cells, making them immunologically similar to patients with acquired immunodeficiency syndrome (AIDS). Both patients with AIDS and Aalpha0/0 mice have hypertrophic gastric folds. To clarify the mechanism of gastric mucosal hyperplasia, we investigated the pathophysiology and the role of the innate immunity in the stomach of Aalpha0/0 mice. METHODS: Stomachs from 1-6 month old Aalpha0/0 mice, kept under specific pathogen free conditions, were examined at 1 month intervals histologically and immunohistochemically. Gene expression of proinflammatory cytokines, Toll-like receptors (TLRs), cyclooxygenase (COX)-2, and myeloperoxidase (MPO) activity in the gastric mucosa was investigated. Serum gastrin levels and gastric acidity were measured. Bacterial culture of the stomach was performed. To clarify the roles of hypergastrinaemia in the gastric mucosa, a gastrin receptor antagonist (AG041R) was administered. RESULTS: Aalpha0/0 mice had a diffusely thick corpus mucosa with infiltration of CD11b+ granulocytes and macrophages. Anti-Ki67 staining demonstrated expansion of the proliferating neck zone. Gene expression of interleukin 1beta, interferon gamma, TLR-2, TLR-4, and COX-2 were upregulated, and MPO activity was increased. Only a small amount of non-pathogenic bacteria was detected in the stomach. Serum gastrin levels and Reg-Ialpha positive cells in the gastric mucosa increased, despite normal gastric acidity. After treatment with AG041R, gastric mucosal thickness was significantly reduced. CONCLUSION: Persistent activation of innate immunity in the stomach induced gastric mucosal hyperplasia through upregulation of gastrin synthesis in Aalpha0/0 mice, suggesting a pathophysiology similar to the gastric changes in patients with AIDS.

Identification of a gene element essential for leukemia-specific expression of transgenes.

Leukemia-specific promoters and enhancers for gene therapy had never been reported. Since the Wilms' tumor gene WT1 is overexpressed in almost all types of leukemia, WT1 is an ideal target of leukemia-specific therapy. To explore the possibility of gene therapy for leukemia using WT1 promoter and enhancer, their activities in several kinds of cells were analyzed by using the enhanced green fluorescent protein (EGFP) gene as a reporter. First, we identified the best combination (654P/EGFP/int3- enh/3'-enh vector) of the 654-bp WT1 promoter and the two WT1 enhancers located in intron 3 and at the 3' end of the WT1 gene for inducing EGFP expression in K562 cells, which endogenously expressed WT1. When this was transfected into WT1-expressing leukemia cells (K562, HEL), WT1-nonexpressing hematopoietic cells (Daudi, U937), and WT1-expressing nonhematopoietic cells (TYK-nu-CPr, SW480, 293 T), 19.8, 22.9, 1.47, 1.43, 4.50, 4.16, and 1.09 times EGFP expression was induced, respectively, compared to that by the promoter-less EGFP vector. These results showed that the 654P/EGFP/int3-enh/3'-enh vector specifically induced high levels of EGFP expression in WT1-expressing leukemia cells. 654P/int3- enh/3'-enh vector containing transgenes such as suicide genes might become useful tools for leukemia-specific gene therapy.

Inhibitory effects of Helicobacter pylori infection on murine autoimmune gastritis.

BACKGROUND AND AIM: Long term Helicobacter pylori infection leads to atrophic gastritis but the relation between H pylori infection and autoimmune related atrophic gastritis (AIG) remains unclear. We studied the effects of H pylori infection on the pathophysiology of AIG in mice. MATERIALS AND METHODS: BALB/c nu/nu mice (n=40) with or without H pylori infection received splenocytes from neonatally thymectomised mice to induce AIG. Half of the mice were orally infected with H pylori prior to AIG induction. Histological findings, and local and systemic immune responses were serially evaluated. RESULTS: Two and six months after transfer, parietal cells in uninfected mice were depleted while those in infected mice were well preserved. The degree of gland atrophy (p<0.01), hyperplasia (p<0.01), gastric pH (p<0.05), and serum gastrin levels of infected mice were significantly lower than those of uninfected mice. Serum antiparietal cell antibody levels gradually decreased in infected mice, and were significantly lower than those of uninfected mice at six months (p<0.05). Real time polymerase chain reaction studies revealed significantly higher interleukin 4 (p<0.05) and transforming growth factor beta (p<0.05) gene expression in the gastric mucosa in infected mice than in uninfected mice at both two and six months after AIG induction. CONCLUSIONS: H pylori infection inhibited the development of AIG in mice. Th2-type immune responses and transforming growth factor beta in the gastric microenvironment might be involved in the inhibitory effects of H pylori infection on the development of AIG, in which Th1-type responses have an important role.

[Anesthetic management for cardioverter defibrillator implantation in a patient with preoperatively undiagnosed myotonic dystrophy].

We unexpectedly anesthetized a 31-year-old male with mild myotonic dystrophy (MD) that had not been diagnosed preoperatively, for implantable cardioverter defibrillator (ICD) implantation. Although MD is an uncommon disorder, cardiac conduction abnormalities and dilated cardiomyopathy are seen commonly in these patients. Therefore, chances of ICD implantation may increase in MD patients. But, the patients are often unaware of this disease and it is common for them to conceal their symptoms, and the diagnosis may not be made before the operation. We emphasize that preoperative assessment is important and that it is necessary to consider MD in patients for ICD implantation.

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells.

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.

Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes.

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.

Metastin suppresses the motility and growth of CHO cells transfected with its receptor.

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.