Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.     

Inhibition of endogenous BMP in the glomerulus leads to mesangial matrix expansion

Bone morphogenetic protein7 (BMP7) attenuates renal tubular and interstitial damage in a variety of experimental models. The function of BMP in the glomerulus is, however, not well understood. In the present study, we generated transgenic mice carrying cDNA for noggin, an endogenous inhibitor of BMPs, driven by the podocyte-specific promoter nephrin. Transgenic founder mice could be divided into two groups based on gross histological analyses at 2 months of age. One group was characterized by the presence of cystic glomeruli with collapsed capillary tufts and a decrease in mesangial cell number, representing a developmental defect during glomerular morphogenesis ("cystic" Tg mice). In contrast, the kidneys appeared to be normal in the other group ("non-cystic" Tg mice). In both groups, however, massive mesangial expansion developed at 10 months of age. The lesion was characterized by the accumulation of fibronectin, but not type I collagen, type IV collagen or laminin. This phenotype is similar to the fibronectin nephropathy. These results suggest that endogenous BMP can have an important role in regulating glomerular structural homeostasis.     

Embryo outcome in Y-chromosome microdeleted infertile males after ICSI

A prospective study involving 118 infertile Japanese couples to assess the embryo outcomes in both azoospermic and oligoasthenoteratoazoospermic (OAT) patients with Y-chromosome microdeletion. The men were divided into two groups; azoospermia (n = 27), and OAT, sperm concentration <5 x 10(6)/ml (n = 91). They were investigated for Y-chromosome microdeletions by a polymerase chain reaction (PCR) amplification of the Y-chromosome-specific sequence tag site (STS). The embryo outcomes of patients found to have Y-microdeletion were determined. The frequency of microdeletion was 8.8% (9) and two had microdeletions distal to DAZ. The mean fertilization rate and the cleavage rate in the eight cycles of both azoospermic and oligospermic patients were 59.3 and 87.5%, respectively. The percentages of grade 1 & 2 embryos, > or =6 cells embryos, and blastocyts were 51.7, 65.6, and 45.3%, respectively. Three pregnancies resulted from the eight cycles (37.5%). CONCLUSION: in Y-chromosome microdeletion cycles in which sperm cells were available for intracytoplasmic sperm injection (ICSI), embryo outcome was comparable to conventional IVF.     

Interactions between Serum Vitamin D Levels and Vitamin D Receptor Gene FokI Polymorphisms for Renal Function in Patients with Type 2 Diabetes

Background

We aimed to examine associations among serum 25-hydroxyvitamin D (25OHD) levels, 1,25-dihyroxyvitamin D (1,25OHD) levels, vitamin D receptor (VDR) polymorphisms, and renal function based on estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes.

Methods

In a cross-sectional study of 410 patients, chronic kidney disease (CKD) stage assessed by eGFR was compared with 25OHD, 1,25OHD, and VDR FokI (rs10735810) polymorphisms by an ordered logistic regression model adjusted for the following confounders: disease duration, calendar month, use of angiotensin converting enzyme inhibitors/angiotensin receptor blockers or statins, and serum calcium, phosphate, and intact parathyroid hormone levels.

Results

1,25OHD levels, rather than 25OHD levels, showed seasonal oscillations; peak levels were seen from May to October and the lowest levels were seen from December to February. These findings were evident in patients with CKD stage 3∼5 but not stage 1∼2. eGFR was in direct proportion to both 25OHD and 1,25OHD levels (P<0.0001), but it had stronger linearity with 1,25OHD (r = 0.73) than 25OHD (r = 0.22) levels. Using multivariate analysis, 1,25OHD levels (P<0.001), but not 25OHD levels, were negatively associated with CKD stage. Although FokI polymorphisms by themselves showed no significant associations with CKD stage, a significant interaction between 1,25OHD and FokITT was observed (P = 0.008). The positive association between 1,25OHD and eGFR was steeper in FokICT and CC polymorphisms (r = 0.74) than FokITT polymorphisms (r = 0.65).

Conclusions

These results suggest that higher 1,25OHD levels may be associated with better CKD stages in patients with type 2 diabetes and that this association was modified by FokI polymorphisms.     

Changes in Photosynthetic Capacity in Rice Leaves from Emergence through Senescence. Analysis from Ribulose-1,5-bisphosphate Carboxylase and Leaf Conductance

Changes in the rates of gas exchange and the amount of ribulose1,5-bisphosphate (RuBP) carboxylase protein were determinedin the 12th leaf blades of rice during the reproductive stages.RuBP carboxylase exhibited a large change similar to that inthe assimilation rate at 2% O₂ throughout the leaf's life, butits decrease during senescence was barely faster than the decreasein the assimilation rate. Consequently, the overall relationshipwas slightly curvilinear. By contrast, leaf conductance decreasedmore slowly than the assimilation rate which resulted in theintercellular CO₂ concentration increasing during senescence. In order to determine the maximum activity of RuBP carboxylaseat the intercellular CO₂ concentration, the kinetic parametersand their pH response were determined using purified, and completelyactivated, rice RuBP carboxylase. The maximum carboxylase activityat the intercellular CO₂ concentration was linearly correlatedwith the assimilation rate at 2% O₂ (r=0.989), and was veryclose to that needed to account for the assimilation rate. We conclude that changes in both the amount of RuBP carboxylaseprotein and leaf conductance reflect the change in photosynthesisduring the life span of the leaf. (Received November 26, 1983; Accepted February 20, 1984)     

Prostacyclin inhibits 5-hydroxytryptamine release but stimulates thromboxane synthesis during cardiopulmonary bypass

The antiaggregating agent prostacyclin (PGI₂) was infused into ten dogs during cardiopulmonary bypass (CPB) to minimize thrombocytopenia and platelet dysfunction. The animals were anesthetized, placed on mechanical ventilation and underwent thoracotomy. After heparinization with 300 u/kg, animals were assigned to control (n=5) or PGI₂ treated groups (n=5). Thoracotomy and then CPB decreased platelet numbers to below 30, 000/mm³ (p < 0.05) and fibrinogen to less than 150 mg/dl (p < 0.05). PGI₂ at 100 ng/kg·min was infused for the 2 h period of CPB. PGI₂ infusion did not prevent these changes, but did prevent platelet serotonin release. In the control group after CPB, platelet serotonin fell from the baseline value of 1.11 μg/10⁹ to 0.35 μg/10⁹ platelets (p < 0.05). In contrast, PGI₂ treatment resulted in a serotonin increase to 2.27 μg/10⁹ platelets (p < 0.05). Thromboxane B₂ concentrations of platelets and plasma rose during CPB (p < 0.05). Surprisingly, PGI₂ infusion accentuated this rise in platelet and plasma thromboxane B₂ (p < 0.05). These data indicate that during CPB, an infusion of PGI₂: 1) does not prevent thrombocytopenia; 2) increases platelet serotonin uptake despite, 3) an associated rise in platelet and plasma thromboxane B₂.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

Detection and quantitative assessment of a Vibrio cholerae O1 species in several foods by a novel enzyme immunoassay.

A selected antibody enzyme immunoassay (SAEIA) for the general detection of Vibrio cholerae O1 species has been developed using the immunological reagents of a rabbit antiserum specific for V. cholerae O1 classical Inaba 569B and immobilized cell fragments of V. cholerae O1 El Tor 85P6, and beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G as tracer. The SAEIA was specific for V. cholerae O1 species and showed low cross-reaction values to other microorganism species tested including Vibrio parahaemolyticus. The detection limit of the SAEIA was 4,500 cells per assay for all the 13 strains of V. cholerae O1 examined. Quantitative comparison on the growth of the El Tor 85P4 in several foods cultured for 24 hr were studied using the SAEIA. Preceding the experiments, little inhibition of every food homogenate for the measurement of the SAEIA was first demonstrated and then the homogenate was directly used for an assay sample. The interaction of the growth of Escherichia coli to that of V. cholerae O1 in a food was also found to be little under the mixed culturing of both bacteria using the SAEIA.