DDOST mutations identified by whole-exome sequencing are implicated in congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.     

Enzymatic Resolution of Racemic Amino Acids

The present paper is concerned with the availability of the acyl derivatives of lysine for the growth of young rats in the course of studying the enzymatic resolution of dl-lysine with mold acylase. The enzymatic resolution of dl-lysine to optically-active l and d-isomers was carried out in either of the following two ways, namely, the asymmetric hydrolysis of diacetyl-dl-lysine or that of ε-benzoyl-α-acetyl-dl-lysine.

The oral administration of ε-acetyl-l-lysine to rats fed on the lysine-deficient diet supported the growth of young rats at a rate approximately two-thirds of that observed when l-lysine was supplied. ε-Benzoyl-l-lysine proved to be quite ineffective while diacetyl lysine showed a slight but insignificant increase in body weight.     

The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.     

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

High Incidence of Neonatal Danger Signs and Its Implications for Postnatal Care in Ghana: A Cross-Sectional Study

Background

Reducing neonatal mortality is a major public health priority in sub-Saharan Africa. Numerous studies have examined the determinants of neonatal mortality, but few have explored neonatal danger signs which potentially cause morbidity. This study assessed danger signs observed in neonates at birth, determined the correlations of multiple danger signs and complications between neonates and their mothers, and identified factors associated with neonatal danger signs.

Methods

A cross-sectional study was conducted in three sites across Ghana between July and September in 2013. Using two-stage random sampling, we recruited 1,500 pairs of neonates and their mothers who had given birth within the preceding two years. We collected data on their socio-demographic characteristics, utilization of maternal and neonatal health services, and experiences with neonatal danger signs and maternal complications. We calculated the correlations of multiple danger signs and complications between neonates and their mothers, and performed multiple logistic regression analysis to identify factors associated with neonatal danger signs.

Results

More than 25% of the neonates were born with danger signs. At-birth danger signs in neonates were correlated with maternal delivery complications (r = 0.20, p < 0.001), and neonatal complications within the first six weeks of life (r = 0.19, p < 0.001). However, only 29.1% of neonates with danger signs received postnatal care in the first two days, and 52.4% at two weeks of life. In addition to maternal complications during delivery, maternal age less than 20 years, maternal education level lower than secondary school, and fewer than four antenatal care visits significantly predicted neonatal danger signs.

Conclusions

Over a quarter of neonates are born with danger signs. Maternal factors can be used to predict neonatal health condition at birth. Management of maternal health and close medical attention to high-risk neonates are crucial to reduce neonatal morbidity in Ghana.     

Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Alteration of flower colour in <Emphasis Type="Italic">Ipomoea nil</Emphasis> through CRISPR/Cas9-mediated mutagenesis of <Emphasis Type="Italic">carotenoid cleavage dioxygenase 4</Emphasis>

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.