Molecular and physiological analysis of indole-3-acetic acid degradation in Bradyrhizobium japonicum E109

Bradyrhizobium japonicum E109 is a bacterium widely used for inoculants production in Argentina. It is known for its ability to produce several phytohormones and degrade indole-3-acetic acid (IAA). The genome sequence of B. japonicum E109 was recently analyzed and it showed the presence of genes related to the synthesis of IAA by indole-3-acetonitrile, indole-3-acetamide and tryptamine pathways. Nevertheless, B. japonicum E109 is not able to produce IAA and instead has the ability to degrade this hormone under saprophytic culture conditions. This work aimed to study the molecular and physiological features of IAA degradation and identify the genes responsible of this activity. In B. japonicum E109 we identified two sequences coding for a putative 3-phenylpropionate dioxygenase (subunits α and β) responsible for the IAA degradation that were homologous to the canonical cluster of iacC and iacD of Pseudomonas putida 1290. These genes form a separate cluster together with three additional genes with unknown functions. The degradation activity was found to be constitutively expressed in B. japonicum E109. As products of IAA degradation, we identified two compounds, 3-indoleacetic acid 2,3-oxide and 2-(2-hydroperoxy-3-hydroxyindolin-3-yl) acetic acid. Our report proposes, for the first time, a model for IAA degradation in Bradyrhizobium.     

A phage P1 virulent mutation at a new map location

A newly isolated Pl virulent mutation, vir11, is not suppressed by reb mutations that suppress P1virB mutations. The location of P1virll on the vegetative phage map is at the left-most end, beyond all other known markers. Thus, in the circular prophage, virII may be closely linked to the cl repressor gene.     

MYC amplification in angiosarcoma depends on etiological/clinical subgroups – Diagnostic and prognostic value

Angiosarcomas (AS) are rare and heterogeneous malignant vascular tumors with a dismal prognosis. We undertook a retrospective study of AS cases with the intent of elucidating the prevalence of MYC amplification amongst different etiological/clinical subgroups and identifying MYC's potential as a prognostic biomarker.Retrospective collection of data and tumor samples from 110 patients diagnosed with AS in the Netherlands was conducted. Histopathological review and investigation of MYC gene amplification and MYC protein overexpression (using fluorescent in situ hybridization and immunohistochemistry, respectively) was performed.MYC amplification was identified in 50.9 % of cases and predominantly present in secondary AS (sAS) (especially radiotherapy-associated and Stewart-Treves AS, both 92.9 %). Within the primary AS (pAS) subgroup, skin non-UV-associated AS (69.2 %) and primary breast AS (29.4 %) demonstrated MYC amplification. MYC protein overexpression was observed in 47.7 % of all tumors with an overall sensitivity and specificity of 75 % and 81 % respectively. There was no significant difference in median overall survival (OS) between patients with MYC and non-MYC amplified AS. Patients with AS displaying MYC protein overexpression had significantly shorter OS than those without (p = 0.028).Although MYC amplification is characteristic for secondary radiotherapy-associated and Stewart-Treves AS, it is not exclusive to these groups and can also be found in a subset of pAS. Overall concordance of FISH and IHC is rather poor. Only primary breast AS showed 100 % concordance. Therefore MYC expression as surrogate marker should be used with caution. The role of MYC/MYC as a prognostic indicator is undefined, however resulting tailored treatment options could be considered.     

What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3′ end of the core vir regulon. Utilizing the M49 wild type, a mga ^– mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga ^– mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome. Received: 2 May 1996     

Cysteine biosynthesis contributes to β-methylamino-l-alanine tolerance in Escherichia coli

In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin β-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 μg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H₂O₂ in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.     

Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti-CD74 autoantibodies in human ankylosing spondylitis

Ankylosing spondylitis (AS) is associated with autoantibody production to class II MHC-associated invariant chain peptide, CD74/CLIP. In this study, we considered that anti-CD74/CLIP autoantibodies present in sera from AS might recognize CD74 degradation products that accumulate upon deficiency of the enzyme signal peptide peptidase-like 2A (SPPL2a). We analyzed monocytes from healthy controls (n = 42), psoriatic arthritis (n = 25), rheumatoid arthritis (n = 16), and AS patients (n = 15) for SPPL2a enzyme activity and complemented the experiments using SPPL2a-sufficient and -deficient THP-1 cells. We found defects in SPPL2a function and CD74 processing in a subset of AS patients, which culminated in CD74 and HLA class II display at the cell surface. These findings were verified in SPPL2a-deficient THP-1 cells, which showed expedited expression of MHC class II, total CD74 and CD74 N-terminal degradation products at the plasma membrane upon receipt of an inflammatory trigger. Furthermore, we observed that IgG anti-CD74/CLIP autoantibodies recognize CD74 N-terminal degradation products that accumulate upon SPPL2a defect. In conclusion, reduced activity of SPPL2a protease in monocytes from AS predisposes to endosomal accumulation of CD74 and CD74 N-terminal fragments, which, upon IFN-γ-exposure, is deposited at the plasma membrane and can be recognized by anti-CD74/CLIP autoantibodies.     

Physical mapping of the satellite phage P4 genome

Marker-rescue experiments with λ-P4 hybrid phages constructed in vitro have identified the locations of two P4 genes: the transactivation gene (δ) is in the left 10% of the genome, and the DNA replication gene (α) lies between 10 and 70% from the left end of the P4 genome. A λ-P4 hybrid phage which carries all of the essential genes of λ and the left 70% of the P4 genome can produce a P4-like phage which is indistinguishable in phenotype from the parental P4 vir1 phage. One of these P4-like phages has a P4-sized genome which is 70% homologous to P4 and produces λ-specific RNA. The existence of this phage and of two P4 deletion mutants suggests that the right 30% of the P4 genome contains no essential genes.     

PreS deletion profiles of hepatitis B virus (HBV) are associated with clinical presentations of chronic HBV infection

BackgroundThe association of hepatitis B virus (HBV) preS1 and preS2 deletions with progressive liver diseases are not fully understood.ObjectiveThe study aimed to investigate characteristics of HBV preS deletion in HBV-infected patients with different illness categories.Study designTotal of 539 HBV-infected patients were enrolled in the study, including 146 with chronic hepatitis B (CHB), 111 with HBV-related liver cirrhosis (LC), 146 with HBV-related acute-on-chronic liver failure (ACLF), and 136 with HBV-related hepatocellular carcinoma (HCC). PreS deletion was determined by sequencing. Replicons containing representative preS1 and preS2 deletion mutants and wild-type were respectively constructed and transfected into HepG2 cells for phenotypic analysis.ResultsThe detection rates of overall preS deletion were 15.8%, 26.1%, 24.0%, and 34.6% in CHB, LC, ACLF, and HCC patients, respectively. PreS1 deletion was most frequently detected in LC patients while preS2 deletion was most frequently detected in HCC patients, both frequencies were significantly higher than that in CHB patients (17.1% vs. 4.8%, P < 0.01; 19.1% vs. 4.8%, P < 0.01). The deletion patterns across preS gene were different among the 4 illness categories. Compared with wild-type strain, the preS1 deletion mutant had defected preS1 expression, significantly decreased viral mRNA level and SP II promoter activity; while preS2 deletion mutant had defected preS2 expression, and significantly decreased viral mRNA level.ConclusionsHBV preS deletion was associated with advancement of liver diseases not only presented in preS deletion incidence, but also in the deletion pattern. Patients with preS2 deletion might have a higher risk to develop HCC.     

Leucodystrophy and oculocutaneous albinism in a child with an 11q14 deletion

We report a patient with an undetermined leucodystrophy associated with type 1A oculocutaneous albinism (OCA). Type 1 OCA results from recessive mutations in the tyrosinase gene (TYR) located in 11q14.3. The patient was found by FISH to carry a deletion of at least the first exon of the TYR gene on one chromosome and a (TG) deletion at codon 244/245 on the second chromosome. The existence of the microdeletion suggested that a gene responsible for leucodystrophy was located in the vicinity of the TYR gene. A combination of a test of hemizygosity and contig mapping studies allowed us to map the gene within a 0.6 cM region flanked by microsatellite markers D11S1780 and D11S931.


Keywords: leucodystrophy; oculocutaneous albinism; 11q14 deletion; physical mapping     

Maternal–fetal metabolic gene–gene interactions and risk of neural tube defects

Single-gene analyses indicate that maternal genes associated with metabolic conditions (e.g., obesity) may influence the risk of neural tube defects (NTDs). However, to our knowledge, there have been no assessments of maternal–fetal metabolic gene–gene interactions and NTDs. We investigated 23 single nucleotide polymorphisms among 7 maternal metabolic genes (ADRB3, ENPP1, FTO, LEP, PPARG, PPARGC1A, and TCF7L2) and 2 fetal metabolic genes (SLC2A2 and UCP2). Samples were obtained from 737 NTD case-parent triads included in the National Birth Defects Prevention Study for birth years 1999–2007. We used a 2-step approach to evaluate maternal–fetal gene–gene interactions. First, a case-only approach was applied to screen all potential maternal and fetal interactions (n = 76), as this design provides greater power in the assessment of gene–gene interactions compared to other approaches. Specifically, ordinal logistic regression was used to calculate the odds ratio (OR) and 95% confidence interval (CI) for each maternal–fetal gene–gene interaction, assuming a log-additive model of inheritance. Due to the number of comparisons, we calculated a corrected p-value (q-value) using the false discovery rate. Second, we confirmed all statistically significant interactions (q < 0.05) using a log-linear approach among case-parent triads. In step 1, there were 5 maternal–fetal gene–gene interactions with q < 0.05. The “top hit” was an interaction between maternal ENPP1 rs1044498 and fetal SLC2A2 rs6785233 (interaction OR = 3.65, 95% CI: 2.32–5.74, p = 2.09 × 10^− 8, q = 0.001), which was confirmed in step 2 (p = 0.00004). Our findings suggest that maternal metabolic genes associated with hyperglycemia and insulin resistance and fetal metabolic genes involved in glucose homeostasis may interact to increase the risk of NTDs.     

Effects of Azospirillum brasilense with genetically modified auxin biosynthesis gene ipdC upon the diversity of the indigenous microbiota of the wheat rhizosphere

The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.     

Clinical significance of the reduction of UT-B expression in urothelial carcinoma of the bladder

Urea transporter B (UT-B) is a membrane protein and plays an important role in regulating urea concentration in bladder urothelial cells. It has been reported that UT-B gene mutations were related to bladder carcinogenesis, and UT-B deletion could induce DNA damage and apoptosis in bladder urothelium. However, the functions and clinical significance of UT-B in human bladder cancer remain unknown. The most common type of bladder cancer is urothelial carcinoma (UC). We hypothesized that UT-B expression was related to bladder UC progress. In this study, UT-B was detected using immunohistochemistry in 52 paraffin-embedded specimens of bladder UC and 10 normal urothelium specimens. The results showed that UT-B protein expression in UC tumor cells was significantly lower as compared with normal urothelial cells (P = 0.021). UT-B protein expression was significantly reduced with increasing histological grade (P = 0.010). UT-B protein expression in muscle-invasive stage was significantly lower than in non-muscle-invasive stage (P = 0.014). Taken together, our data suggest that the reduction or loss of UT-B expression may be related to the incidence, progression and invasiveness of bladder UC. UT-B may be a novel diagnostic or prognostic biomarker, as well as a potential therapeutic target in UC of the bladder.     

Novel intragenic deletions within the <Emphasis Type="Italic">UBE3A</Emphasis> gene in two unrelated patients with Angelman syndrome: case report and review of the literature

Background

Patients with Angelman syndrome (AS) are affected by severe intellectual disability with absence of speech, distinctive dysmorphic craniofacial features, ataxia and a characteristic behavioral phenotype. AS is caused by the lack of expression in neurons of the UBE3A gene, which is located in the 15q11.2-q13 imprinted region. Functional loss of UBE3A is due to 15q11.2-q13 deletion, mutations in the UBE3A gene, paternal uniparental disomy and genomic imprinting defects.

Case presentation

We report here two patients with clinical features of AS referred to our hospital for clinical follow-up and genetic diagnosis. Methylation Specific-Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) of the 15q11.2-q13 region was carried out in our laboratory as the first diagnostic tool detecting two novel UBE3A intragenic deletions. Subsequently, the MLPA P336-A2 kit was used to confirm and determine the size of the UBE3A deletion in the two patients. A review of the clinical features of previously reported patients with whole UBE3A gene or partial intragenic deletions is presented here together with these two new patients.

Conclusion

Although rare, UBE3A intragenic deletions may represent a small fraction of AS patients without a genetic diagnosis. Testing for UBE3A intragenic exonic deletions should be performed in those AS patients with a normal methylation pattern and no mutations in the UBE3A gene.

     

A novel deletion of SNURF/SNRPN exon 1 in a patient with Prader-Willi-like phenotype

Here we report the smallest deletion involving SNURF/SNRPN that causes major symptoms of Prader-Willi syndrome (PWS), including hypotonia, dysmorphic features, intellectual disability, and obesity. A female patient with the aforementioned and additional features was referred to the Mayo Clinic Cytogenetics laboratory for genetic testing. Chromosomal microarray analysis and subsequent Sanger sequencing identified a de novo 6.4 kb deletion at 15q11.2, containing exon 1 of the SNURF gene and exon 1 of the shortest isoform of the SNRPN gene. SNURF/SNRPN exon 1, which is methylated on the silent maternal allele, is associated with acetylated histones on the expressed paternal allele. This region also overlaps with the PWS-imprinting center (IC). Subsequent molecular methylation analysis was performed using methylation-specific MLPA (MS-MLPA), which characterized that the deletion of SNURF/SNRPN exon 1 was paternal in origin, consistent with the PWS-like phenotype. Since SNURF/SNRPN gene and the PWS-IC are known to regulate snoRNAs, it is likely that the PWS-like phenotype observed in patients with paternal SNURF/SNRPN deletion is due to the disrupted expression of SNORD116 snoRNAs.     

Desmin splice variants causing cardiac and skeletal myopathy

Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A→G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G→A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A→G and IVS2-1G→A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.


Keywords: cardiac and skeletal myopathy; desmin splice site mutations; expression study; genotype-phenotype correlation