Loeys–Dietz syndrome type 4, caused by chromothripsis,involving the TGFB2 gene

The TGF-β signaling pathway controls cellular proliferation, growth and differentiation and regulates several functions of the connective tissue. Disruption of genes coding for components of the TGF-β signaling pathway or its interactors, such as fibrillin-1, has been shown to cause several human pathologies. Large deletions and non-sense mutations in TGFB2 gene have been recently described in patients with aortic aneurysm, scoliosis, arachnodactyly, chest deformities, joint hyper-flexibility, and mild intellectual disability; this condition has been called Loeys–Dietz syndrome, type 4. In this paper we describe an 18-year-old girl with borderline mental impairment, seizures, retinal degeneration, short stature, congenital hip dysplasia, severe and worsening joint hypermobility, scoliosis, progressive deformation of the long bones, aortic dilatation and platelet disorder. Molecular study of DNA by Array-CGH demonstrated four de novo microdeletions: TGFB2 is among the genes deleted and we consider it the obvious candidate for the clinical phenotype. The multiple chromosomal rearrangements detected in the current patient can be ascribed to an event of constitutional chromothripsis.     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

The first Japanese case of the arthrochalasia type of Ehlers–Danlos syndrome with COL1A2 gene mutation

This is the first report for a Japanese case of arthrochalasia type of Ehlers–Danlos syndrome (EDS). A 46-year-old woman consulted us for joint hypermobility and skin hyperextensibility that had been present soon after birth. There was no family history of a similar disease. She was diagnosed as having bilateral congenital hip dislocation and bilateral habitual shoulder dislocation at her childhood. Her skin was velvety, doughy and hyperextensible. She showed hypermobility of the joints of the hands and feet and generalized joint laxity, with no evidence of scoliosis. Electrophoretic analysis of collagenous proteins revealed the presence of an additional band in the position of pNα2(I) in the sample from culture medium of the patient fibroblasts. Analysis of the α2 chains of type I collagen gene, COL1A2, showed a heterozygous G to T transition at the + 1 position of the exon 6 donor splice site (c.279+1G>T). This mutation resulted in skipping of exon 6, which leads to deficient processing of the amino-terminal end of proα2(I) chains of type I collagen. Based on these findings, we made a diagnosis of the arthrochalasia type of EDS, which corresponds to EDS type VIIB in the former classification.     

Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in Streptomyces virginiae

Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M₁ and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW ₂ located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae. An erratum to this article can be found at     

Identification,characterization, and crystal structure of an aldo–keto reductase (AKR2E4) from the silkworm Bombyx mori

A new member of the aldo–keto reductase (AKR) superfamily with 3-dehydroecdysone reductase activity was found in the silkworm Bombyx mori upon induction by the insecticide diazinon. The amino acid sequence showed that this enzyme belongs to the AKR2 family, and the protein was assigned the systematic name AKR2E4. In this study, recombinant AKR2E4 was expressed, purified to near homogeneity, and kinetically characterized. Additionally, its ternary structure in complex with NADP⁺ and citrate was refined at 1.3 Å resolution to elucidate substrate binding and catalysis. The enzyme is a 33-kDa monomer and reduces dicarbonyl compounds such as isatin and 17α-hydroxy progesterone using NADPH as a cosubstrate. No NADH-dependent activity was detected. Robust activity toward the substrate inhibitor 3-dehydroecdysone was observed, which suggests that this enzyme plays a role in regulation of the important molting hormone ecdysone. This structure constitutes the first insect AKR structure determined. Bound NADPH is located at the center of the TIM- or (β/α)₈-barrel, and residues involved in catalysis are conserved.     

The complete intron/exon structure of Ephydatia mülleri fibrillar collagen gene suggests a mechanism for the evolution of an ancestral gene module

We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5 junction exon and the two last 3 exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.Abbreviations used bp base pair(s) - kb kilobase(s) - C-protease the enzyme that cleaves the carboxyl-terminal propeptide     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Binding and Cleavage of E. coli HUβ by the E. coli Lon Protease

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUβ, HUα, or HUβ-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUβ.     

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3′-hydroxylase gene

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3′H, the gene for flavonoid 3′-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.     

A rapid and simple electrophoretic method for the detection of mutations involving small insertion or deletion: application to β-thalassemia

Summary The 1.8-kb -globin gene fragments of DNAs from individuals heterozygous for nine different -thalassemia mutations involving 1, 2, 3, 4, or 25 basepair (bp) insertions or deletions were amplified by the polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on aqueous 8% polyacrylamide gel. In each heterozygote with either a 2 to 25 bp deletion, but not with a 1 bp insertion, two slower migrating bands representing heteroduplexes in addition to the 1.8-kb homoduplex band were seen. The electrophoretic positions of these slower migrating bands were characteristic of each mutation studied. By co-amplification with known normal DNA, it was also possible to distinguish DNAs from normal individuals and from individuals who are homozygous for the small insertion/deletion mutations. These studies demonstrate that the heteroduplex formation generated in PCR can be applied as a simple method in the diagnosis of insertion/deletion mutations involving 2 to 25 bp in -thalassemias as well as in other genetic disorders.     

Identification of novel mutations in the ABCA12 gene,c.1857delA and c.5653–5655delTAT,causing harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe autosomal recessive developmental disorder of the skin that is frequently but not always fatal in the first few days of life. In HI, mutations in both ABCA12 gene alleles must have a severe impact on protein function and most mutations are truncating. The presence of at least one nontruncating mutation (predicting a residual protein function) usually causes a less severe congenital ichthyosis (lamellar ichthyosis or congenital ichthyosiform erythroderma). Here we report on a girl with severe HI diagnosed by prenatal ultrasound at 33 5/7 week gestation. Ultrasound findings included ectropion, eclabium, deformed nose, hands and feet, joint contractures, hyperechogenic amniotic fluid and polyhydramnion. After birth, palliative treatment was provided and she died on her first day of life. Sequence analysis of the ABCA12 gene identified two novel mutations, c.1857delA (predicting p.Lys619*) in exon 15 and c.5653–5655delTAT (predicting p.1885delTyr) in exon 37, each in heterozygous state. The c.5653–5655delTAT mutation is not truncating, but the deleted tyrosine at position 1885 is perfectly conserved among vertebrates and molecular studies evaluated the mutation as probably disease causing and damaging.     

Genetic and phytochemical analysis of the in vitro regenerated Pilosocereus robinii by ISSR,SDS–PAGE and HPLC

Pilosocereus robinii is a rare species which is experiencing sudden population collapse. Identifying and developing effective conservation and management strategies to halt the forestall extinction of this species is crucial. The present study was conducted to assess the best conditions for in vitro propagation of this plant in regard to its morphogenic, genetic as well as the chemical potentials. A successful in vitro propagation system of P. robinii has been developed. MS hormone-free medium induced the best root morphogenic potential. The plants were acclimatized in the greenhouse at 100% survival rate. Besides, the somaclonal variations between the in vitro raised plants were analyzed using PCR-ISSR markers and SDS–PAGE protein, where the regenerated explants on MS medium supplemented with TDZ were the highest in inducing new specific marker bands. Sh6 ISSR primer showed the highest polymorphism value, 81.8% with 33 total amplified fragments, while Sh3 ISSR primer showed the lowest value with polymorphic percentage of 14.3%. Furthermore, SDS–PAGE protein analysis showed no variation in protein pattern of the studied treatments. On the other side, HPLC analysis of the in vitro plantlets extracts has shown that 2iP based treatments were the highest in organic acids accumulation, while the phenolic constituents' accumulation was found to reach its peak in the BA based treatments.     

A structure–activity relationship study of flavonoids as inhibitors of E. coli by membrane interaction effect

Flavonoids exhibit a broad range of biological activities including antibacterial activity. However, the mechanism of their antibacterial activity has not been fully investigated. The antibacterial activity and membrane interaction of 11 flavonoids (including 2 polymethoxyflavones and 4 isoflavonoids) against Escherichia coli were examined in this study. The antibacterial capacity was determined as flavonoids > polymethoxyflavones > isoflavonoids. Using fluorescence, it was observed that the 5 flavonoids rigidified the liposomal membrane, while the polymethoxyflavones and isoflavonoids increased membrane fluidity. There was a significant positive correlation between antibacterial capacity and membrane rigidification effect of the flavonoids. A quantitative structure–activity relationship (QSAR) study demonstrated that the activity of the flavonoid compounds can be related to molecular hydrophobicity (CLogP) and charges on C atom at position3 (C3). The QSAR model could be used to predict the antibacterial activity of flavonoids which could lead to natural compounds having important use in food and medical industry.     

Leishmania major: Anti-leishmanial activity of Nuphar lutea extract mediated by the activation of transcription factor NF-κB

Here we report the effect of a partially purified alkaloid fraction (NUP) of Nuphar lutea on nuclear factor kappa B (NF-κB) expression and studied its mechanism of toxicity against Leishmania major in C3H mice peritoneal macrophages. NUP was found to be a mixture of thermo-stable dimeric sesquiterpene thioalkaloids containing mainly thionupharidines. The anti-leishmanial activity was shown to be mediated through the activation of NF-κB and increased iNOS production. Additionally, the nitric oxide inhibitor, N^G-monomethyl-l-arginine (0.5 mM) totally reverted the anti-leishmanial effect of NUP (0.25 and 0.5 μg/ml). NUP was also shown to act as an anti-oxidant, almost completely inhibiting the macrophage respiratory burst activity. However, no elevated lysozyme (EC3.2.1.17) or β-galactosidase (EC3.2.1.23) activities were demonstrated in macrophages treated with NUP. This study suggests, that the activity of NUP is mediated by NF-κB activation and the production of nitric oxide which is dependent on the l-arginine:NO pathway.