Insulin is imprinted in the placenta of the marsupial, Macropus eugenii

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.     

Activation of prothrombin by ASP, a serine protease released from Aeromonas sobria

The effect of a serine protease (ASP) secreted from Aeromonas sobria on plasma coagulation was investigated. Proteolytically active ASP promoted human plasma coagulation in a dose-dependent manner. Consistent with the preference for a factor Xa-specific oligo-peptide substrate, ASP produced enzymatic activity from human prothrombin but not from factors IX and X. ASP cleaved prothrombin to produce enzymatically active 37 kDa-fragment displaying the same molecular mass as alpha-thrombin. ASP is the first bacterial serine protease that produces alpha-thrombin, through which ASP may contribute to the induction of thrombotic tendency in disseminated intravascular coagulation complicated with sepsis caused by A. sobria infections.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.     

Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.     

Efficacy and Safety of Sitagliptin Added to Insulin in Japanese Patients with Type 2 Diabetes: The EDIT Randomized Trial

Aims

To clarify the efficacy and safety of adding sitagliptin to insulin therapy in Japanese patients with suboptimally controlled type 2 diabetes (T2DM).

Study Design and Methods

This was a 24-week, prospective, randomized, open-labeled, controlled trial. Patients with T2DM who were suboptimally controlled despite receiving at least twice daily injection of insulin were enrolled in the study. The patients were randomized to continuation of insulin treatment (Insulin group) or addition of sitagliptin 50 to 100 mg daily to insulin treatment (Ins+Sita group). The primary outcome was change in HbA1c at week 24.

Results

Adding sitagliptin to insulin significantly reduced HbA1c from 7.9 ± 1.0% at baseline to 7.0 ± 0.8% at week 24 (P <0.0001), while there was no significant change in HbA1c in the Insulin group (7.8 ± 0.7% vs. 7.8 ± 1.1%, P = 0.32). The difference in HbA1c reduction between the groups was 0.9% (95% confidence interval, 0.4 to 1.5, P = 0.01). There was no significant weight gain in either group. Incidence of hypoglycemia was significantly reduced in the Ins+Sita group compared with the Insulin group. Treatment satisfaction was improved in the Ins+Sita group. Baseline HbA1c level and beta cell function were associated with the magnitude of reduction in HbA1c in the Ins+Sita group.

Conclusion

Adding sitagliptin to insulin reduced HbA1c without weight gain or increase in hypoglycemia, and improved treatment satisfaction in Japanese patients with T2DM who were suboptimally controlled despite at least twice daily injection of insulin.

Trial Registration

The University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000004678     

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.     

Heparan sulfate is required for bone morphogenetic protein-7 signaling

Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Analysis of the microheterogeneity of the glycan chain of rat transferrin

To investigate the microheterogeneity of the glycan chain of rat transferrin, either the protein moiety was labeled with 125I or the sialyl residues with 3H. The molecule was then subjected to Con A chromatography. Three components were obtained. Each was enzymatically desialylated and sialyl/protein molar ratios were calculated. The native protein as well as the 3 components were also subjected to isoelectric focusing. The results indicated that rat transferrin may have 3 types of glycan chain: The major type (60%) corresponds to a molecular species with triantennary branching, while 30% consists of molecules with biantennary and 10% with tetraantennary branching. The last species has not been previously described.