Highly polymorphic repeat marker within the β-amyloid precursor protein gene

We have identified a polymorphic compound dinucleotide repeat sequence in intron 1 of the -amyloid precursor protein (APP) gene on chromosome 21. Using polymerase chain reaction (PCR) amplification of the locus, designated APPivsl, we detected 13 alleles in the CEPH family members (heterozygosity = 0.69). Lod score analysis showed complete linkage of the marker to the loci D21S210 and D21221.     

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.     

Ultrastructure of the differentiating zygotospores of Porphyra leucosticta (Rhodophyta)

The ultrastructure of zygotosporogenesis is described for the red alga Porphyra leucosticta Thuret. Packets of eight zygotosporangia, each packet derived from a single carpogonium are interspersed among vegetative cells. Zygotospore differentiation in Porphyra can be separated into three developmental stages. (i) Young zygotospores exhibit a nucleus and a large centrally located, lobed plastid with pyrenoid. Mucilage is produced within concentric membrane structures during their dilation, thus resulting in the formation of mucilage sacs. Subsequently, these sacs release their contents, initiating the zygotospore wall formation. Straight‐profiled dictyosomes produce vesicles that also provide wall material. During the later stages of young zygotospores, starch polymerization commences, (ii) Medium‐aged zygotospores are characterized by the presence of fibrous vacuoles. These are formed from the ‘fibrous vacuole associated organelles’. The fibrous vacuoles finally discharge their contents. (iii) Mature zygotospores are recognized by the presence of numerous cored vesicles produced by dictyosomes. Cored vesicles either discharge their contents or are incorporated into the fibrous vacuoles. There is a gradual reduction of starch granules during zygotospore differentiation. Mature zygotospores are surrounded by a fibrous wall, have a large chloroplast with pyrenoid and well‐depicted phycobilisomes but are devoid of starch granules.     

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.Marie Wattenhofer and Nilüfer Sahin-Calapoglu contributed equally to this work     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Conserved non-genic sequences - an unexpected feature of mammalian genomes

Mammalian genomes contain highly conserved sequences that are not functionally transcribed. These sequences are single copy and comprise approximately 1-2% of the human genome. Evolutionary analysis strongly supports their functional conservation, although their potentially diverse, functional attributes remain unknown. It is likely that genomic variation in conserved non-genic sequences is associated with phenotypic variability and human disorders. So how might their function and contribution to human disorders be examined?     

Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.     

Isolation and Initial Characterization of a Novel Zinc Finger Gene, DNMT3L, on 21q22.3, Related to the Cytosine-5- Methyltransferase 3 Gene Family

We have isolated the DNMT3L gene that is related to the cytosine-5-methyltransferase 3 (DNMT3) family. The gene is located on chromosome 21q22.3 between the AIRE and the KIAA0653 genes and spans approximately 16 kb of genomic sequence. The encoded protein of 387 amino acids has a cysteine-rich region containing a novel-type zinc finger domain that is conserved in DNMT3A and DNMT3B but also in ATRX, a member of the SNF2 protein family. The novel domain, called an ADD (ATRX, DNMT3, DNMT3L)-type zinc finger, contains two subparts: a C2C2 and an imperfect PHD zinc finger. Expression of the DNMT3L mRNA was not detectable by Northern blotting; however, RT-PCR amplification revealed that it is expressed at low levels in several tissues including testis, ovary, and thymus.     

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.