Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH cytochrome b5 reductase

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

Polyploid measles virus with hexameric genome length

Particles of most virus species accurately package a single genome, but there are indications that the pleomorphic particles of parainfluenza viruses incorporate multiple genomes. We characterized a stable measles virus mutant that efficiently packages at least two genomes. The first genome is recombinant and codes for a defective attachment protein with an appended domain interfering with fusion-support function. The second has one adenosine insertion in a purine run that interrupts translation of the appended domain and restores function. In that genome, a one base deletion in a different purine run abolishes polymerase synthesis, but restores hexameric genome length, thus ensuring accurate RNA encapsidation, which is necessary for efficient replication. Thus, the two genomes are complementary. The infection kinetics of this mutant indicate that packaging of multiple genomes does not negatively affect growth. We also show that polyploid particles are produced in standard infections at no expense to infectivity. Our results illustrate how the particles of parainfluenza viruses efficiently accommodate cargoes of different volume, and suggest a mechanism by which segmented genomes may have evolved.

SOCS3 regulates onset and maintenance of TH2- mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (TH2) cells, but its role in TH2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased TH2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased TH2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of TH2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.

Peripheral markers of the gamma-aminobutyric acid (GABA)ergic system in Angelman's syndrome.

It has recently been demonstrated that patients with Angelman's syndrome who exhibited a deletion on cytogenetic tests show more severe clinical pictures with drug-resistant epilepsy than patients with Angelman's syndrome not carrying the deletion. To verify if this difference in clinical severity can be attributed to genes for the three gamma-aminobutyric acid (GABA)A receptor subunits (GABRB3, GABRA5, GABRG3) located in the deleted region, a possible modification of peripheral markers of the GABAergic system was investigated in 12 subjects with Angelman's syndrome and 20 age-matched subjects (8 with idiopathic epilepsy and 12 not affected by neurologic diseases). The results confirmed a more severe clinical picture, and epilepsy syndrome in particular, in Angelman's syndrome patients with deletions versus patients without deletions. In contrast, biochemical study (based on dosage of plasma levels of GABA and diazepam binding inhibitor, an endogenous ligand of GABAA and peripheral benzodiazepine receptors, showed contradictory results: patients with Angelman's syndrome showed significantly higher levels of GABA and diazepam binding inhibitor than patients without neurologic impairment but significantly lower levels than epileptic controls.

Lack of MEF2A Δ7aa mutation in Irish families with early onset ischaemic heart disease, a family based study

Background: Ischaemic heart disease (IHD) is a complex disease due to the combination of environmental and genetic factors. Mutations in the MEF2A gene have recently been reported in patients with IHD. In particular, a 21 base pair deletion (Δ7aa) in the MEF2A gene was identified in a family with an autosomal dominant pattern of inheritance of IHD. We investigated this region of the MEF2A gene using an Irish family-based study, where affected individuals had early-onset IHD. Methods: A total of 1494 individuals from 580 families were included (800 discordant sib-pairs and 64 parent-child trios). The Δ7aa region of the MEF2A gene was investigated based on amplicon size. Results: The Δ7aa mutation was not detected in any individual. Variation in the number of CAG (glutamate) and CCG (proline) residues was detected in a nearby region. However, this was not found to be associated with IHD. Conclusion: The Δ7aa mutation was not detected in any individual within the study population and is unlikely to play a significant role in the development of IHD in Ireland. Using family-based tests of association the number of tri-nucleotide repeats in a nearby region of the MEF2A gene was not associated with IHD in our study group. © 2006 Horan et al; licensee BioMed Central Ltd.

An algorithm to predict pregnancy in assisted conception

BACKGROUND: Male fertility potential cannot be measured by conventional parameters for assisted reproduction by intracytoplasmic sperm injection. This study determines the relationship between testicular and ejaculated sperm mitochondrial (mt) DNA deletions, nuclear (n) DNA fragmentation and fertilisation and pregnancy rates in ICSI. METHODS: Ejaculated sperm were obtained from 77 men and testicular sperm from 28 men with obstructive azoospermia undergoing ICSI. Testicular sperm were retrieved using a Trucut needle. MtDNA analysed using a long polymerase chain reaction. The alkaline Comet assay determined nDNA fragmentation. RESULTS: Of subjects who achieved a pregnancy (50%) using testicular sperm, only 26% had partners�??�?�¢?? sperm with wild type (WT) mtDNA. Of pregnant subjects (38%) using ejaculated sperm, only 8% had partner sperm with WT mtDNA.. In each, the successful group had less mtDNA deletions and less nDNA fragmentation. There were inverse relationships between pregnancy and mtDNA deletion numbers, size and nDNA fragmentation for both testicular and ejaculated sperm. No relationships were observed with fertilisation rates. An algorithm for the prediction of pregnancy is presented based on the quality of sperm nDNA and mtDNA. CONCLUSION: In both testicular and ejaculated sperm, mtDNA deletions and nDNA fragmentation are closely associated with pregnancy in ICSI.

Characterization of the 5' upstream regulatory region of the human myostatin gene: regulation of myostatin gene transcription by dexamethasone in vitro

We cloned and characterized a 3.3-kb fragment containing the 5'-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-kappaB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C(2)C(12) and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.

A reactive force field simulation of liquid-liquid phase transitions in phosphorus

A force field model of phosphorus has been developed based on density functional (DF) computations and experimental results, covering low energy forms of local tetrahedral symmetry and more compact (simple cubic) structures that arise with increasing pressure. Rules tailored to DF data for the addition, deletion, and exchange of covalent bonds allow the system to adapt the bonding configuration to the thermodynamic state. Monte Carlo simulations in the N-P-T ensemble show that the molecular (P-4) liquid phase, stable at low pressure P and relatively low temperature T, transforms to a polymeric (gel) state on increasing either P or T. These phase changes are observed in recent experiments at similar thermodynamic conditions, as shown by the close agreement of computed and measured structure factors in the molecular and polymer phases. The polymeric phase obtained by increasing pressure has a dominant simple cubic character, while the polymer obtained by raising T at moderate pressure is tetrahedral. Comparison with DF results suggests that the latter is a semiconductor, while the cubic form is metallic. The simulations show that the T-induced polymerization is due to the entropy of the configuration of covalent bonds, as in the polymerization transition in sulfur. The transition observed with increasing P is the continuation at high T of the black P to arsenic (A17) structure observed in the solid state, and also corresponds to a semiconductor to metal transition. (C) 2004 American Institute of Physics.

Evaluation of predicted network modules in yeast metabolism using NMR-based metabolite profiling

Genome-scale metabolic models promise important insights into cell function. However, the definition of pathways and functional network modules within these models, and in the biochemical literature in general, is often based on intuitive reasoning. Although mathematical methods have been proposed to identify modules, which are defined as groups of reactions with correlated fluxes, there is a need for experimental verification. We show here that multivariate statistical analysis of the NMR-derived intra- and extracellular metabolite profiles of single-gene deletion mutants in specific metabolic pathways in the yeast Saccharomyces cerevisiae identified outliers whose profiles were markedly different from those of the other mutants in their respective pathways. Application of flux coupling analysis to a metabolic model of this yeast showed that the deleted gene in an outlying mutant encoded an enzyme that was not part of the same functional network module as the other enzymes in the pathway. We suggest that metabolomic methods such as this, which do not require any knowledge of how a gene deletion might perturb the metabolic network, provide an empirical method for validating and ultimately refining the predicted network structure.

A complex zinc finger controls the enzymatic activities of nidovirus helicases.

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells.

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.

Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator.

The elevated levels of beta1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.