A mutation in the gene encoding mitochondrial Mg²+ channel MRS2 results in demyelination in the rat

The rat demyelination (dmy) mutation serves as a unique model system to investigate the maintenance of myelin, because it provokes severe myelin breakdown in the central nervous system (CNS) after normal postnatal completion of myelination. Here, we report the molecular characterization of this mutation and discuss the possible pathomechanisms underlying demyelination. By positional cloning, we found that a G-to-A transition, 177 bp downstream of exon 3 of the Mrs2 (MRS2 magnesium homeostasis factor (Saccharomyces cerevisiae)) gene, generated a novel splice acceptor site which resulted in functional inactivation of the mutant allele. Transgenic rescue with wild-type Mrs2-cDNA validated our findings. Mrs2 encodes an essential component of the major Mg2+ influx system in mitochondria of yeast as well as human cells. We showed that the dmy/dmy rats have major mitochondrial deficits with a markedly elevated lactic acid concentration in the cerebrospinal fluid, a 60% reduction in ATP, and increased numbers of mitochondria in the swollen cytoplasm of oligodendrocytes. MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was dominantly expressed in neurons rather than oligodendrocytes and was ultrastructurally observed in the inner membrane of mitochondria. Our observations led to the conclusion that dmy/dmy rats suffer from a mitochondrial disease and that the maintenance of myelin has a different mechanism from its initial production. They also established that Mg2+ homeostasis in CNS mitochondria is essential for the maintenance of myelin.     

B chromosomes in plants: escapees from the A chromosome genome?

B chromosomes are dispensable elements that do not recombine with the A chromosomes of the regular complement and that follow their own evolutionary track. In some cases, they are known to be nuclear parasites with autonomous modes of inheritance, exploiting "drive" to ensure their survival in populations. Their "selfishness" brings them into conflict with their host nuclear genome and generates a host-parasite relationship, with anti-B-chromosome genes working to ameliorate the worst of their excesses in depriving their hosts of genetic resources. Molecular studies are homing in on their sequence organization to give us an insight into the origin and evolution of these enigmatic chromosomes, which are, with rare exceptions, without active genes.     

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine bound to the A–A mismatch in the CAG/CAG base triad in dsRNA

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine (MCND) connected at the C2 position with a three-atom linker was examined for the binding to mismatches in double stranded RNA. Despite the fully complementary hydrogen bonding groups to guanine, MCND did not bind to guanine–guanine mismatch but did to adenine–adenine mismatch. The base pairs flanking the mismatch had weak effect on the binding, with showing the strongest binding to the A–A mismatch in the CAG/CAG sequence. The A–A mismatch in the GAC/GAC sequence was a poor substrate for the MCND binding. A monomeric derivative of MCND and another derivative lacking a methylcarbamate group showed negligilble binding to the A–A mismatch and the sequence selectivity. These results are important clues for the better molecular design of RNA binding small molecules.     

NR4A nuclear orphan receptors: protective in vascular disease?

PURPOSE OF REVIEW: The nuclear orphan receptors Nur77 (NR4A1), Nurr1 (NR4A2) and NOR-1 (NR4A3) are known to be involved in T-cell apoptosis, brain development, and the hypothalamic-pituitary-adrenal axis. Here, we review our current understanding of the NR4A nuclear receptors in processes that are relevant to vascular disease. RECENT FINDINGS: NR4A nuclear receptors have recently been described to play a role in metabolism by regulating gluconeogenesis, lipolysis, energy expenditure, and adipogenesis. The function of NR4A nuclear receptors has also extensively been investigated in cells crucial in vascular lesion formation, such as macrophages, endothelial cells and smooth muscle cells. SUMMARY: The involvement of NR4A nuclear receptors in both metabolism and in processes in the vessel wall supports a substantial role for NR4A nuclear receptors in the development of vascular disease.     

Latitudinal clines in the grasshopper Dichroplus elongatus: Coevolution of the A genome and B chromosomes?

Argentine populations of Dichroplus elongatus (Orthoptera: Acrididae) are polymorphic for B chromosomes. Previous studies showed that B chromosomes affect body size and some fitness components in Northwestern populations. We studied phenotype and B′s variation patterns along a latitudinal cline as well as the relationship between karyotype and body size related traits in 17 populations from East. Body size related traits showed a ‘saw tooth’ pattern of variation being small at low and high latitudes and large at intermediate latitudes in most of the analysed populations. Analyses of variance and principal components demonstrated that in most analysed populations B carrier males are associated with a decrease in body size related traits with respect to individuals with standard karyotype. Accordingly with the relationship between karyotype and body size, an opposite pattern of latitudinal variation in the frequencies of B′s with respect to body size variation was observed in this area. i.e. smaller individuals tend to have a higher frequency of B chromosomes. The comparison of the differentiation of both karyotype and body size traits with molecular neutral markers demonstrated the relative importance of selection moulding chromosome and phenotype variation. The observed pattern of phenotypic variation is likely to be the result of local adaptation to season length along the latitudinal gradient. The observed contrary pattern of B′s clinal variation may reflect the population ability to maintain this chromosome in relation to the local adaptation. The available evidence indicates that the distribution of B chromosome frequency was shaped by selective factors.     

Caffeine reduces 11β-hydroxysteroid dehydrogenase type 2 expression in human trophoblast cells through the adenosine A(2B) receptor

Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A(2B) receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A(2B) receptor signaling in regulating placental 11β-HSD2, and consequently fetal development.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.     

A Role for NF-κB Activity in Skin Hyperplasia and the Development of Keratoacanthomata in Mice

Background

Previous studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin.

Methods

A knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice) with increased NF-κB activity.

Results

The data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata.

Conclusion

Our findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA) development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced.     

Case–control association study of polymorphisms in the voltage-gated sodium channel genes SCN1A,SCN2A,SCN3A,SCN1B,and SCN2B and epilepsy

High-frequency action potentials are mediated by voltage-gated sodium channels, composed of one large α subunit and two small β subunits, encoded mainly by SCN1A, SCN2A, SCN3A, SCN1B, and SCN2B genes in the brain. These play a key role in epilepsy, with the most commonly mutated gene in epilepsy being SCN1A. We examined whether polymorphisms in the above genes affect epilepsy risk in 1,529 epilepsy patients and 1,935 controls from four ethnicities or locations: Malay, Indian, and Chinese, all from Malaysia, and Chinese from Hong Kong. Of patients, 19 % were idiopathic, 42 % symptomatic, and 40 % cryptogenic. We genotyped 43 polymorphisms: 27 in Hong Kong, 28 in Malaysia, and 12 in both locations. The strongest association with epilepsy was rs3812718, or SCN1A IVS5N+5G>A: odds ratio (OR) = 0.85 for allele G (p = 0.0009) and 0.73 for genotype GG versus AA (p = 0.003). The OR was between 0.76 and 0.87 for all ethnicities. Meta-analysis confirmed the association (OR = 0.81 and p = 0.002 for G, and OR = 0.67 and p = 0.007 for GG versus AA), which appeared particularly strong for Indians and for febrile seizures. Allele G affects splicing and speeds recovery from inactivation. Since SCN1A is preferentially expressed in inhibitory neurons, G may decrease epilepsy risk. SCN1A rs10188577 displayed OR = 1.20 for allele C (p = 0.003); SCN2A rs12467383 had OR = 1.16 for allele A (p = 0.01), and displayed linkage disequilibrium with rs2082366 (r ² = 0.67), whose genotypes tended toward association with SCN2A brain expression (p = 0.10). SCN1A rs2298771 was associated in Indians (OR = 0.56, p = 0.005) and SCN2B rs602594 with idiopathic epilepsy (OR = 0.62, p = 0.002). Therefore, sodium channel polymorphisms are associated with epilepsy.     

A role for the NF-κB pathway in cell protection from complement-dependent cytotoxicity

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKβ, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65(-/-) cells. Furthermore, reconstitution of p65 into p65(-/-) cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65(-/-) cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.     

Mutations in the 5’ NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.     

Vavilosides A1/A2–B1/B2, new furostane glycosides from the bulbs of Allium vavilovii with cytotoxic activity

A phytochemical analysis of the bulbs of Allium vavilovii M. Pop. & Vved. was attained for the first time extensively, affording to the isolation of four new furostanol saponins, named vavilosides A1/A2–B1/B2 (1a/b–2a/2b), as two couple of isomers in equilibrium, together with ascalonicoside A1/A2 (3a/3b) and 22-O-methyl ascalonicoside A1/A2 (4a/4b), previously isolated from shallot, Allium ascalonicum. High concentrations of kaempferol, kaempferide, and kaempferol 4^I-glucoside were also isolated. The chemical structures of the new compounds, established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses, were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-galactopyranosyl 26-O-α-l-rhamnopyranoside (vaviloside A1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-galactopyranosyl 26-O-α-l-rhamnopyranoside (vaviloside A2), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-xylopyranosyl 26-O-α-l-rhamnopyranoside (vaviloside B1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-xylopyranosyl 26-O-α-l-rhamnopyranoside (vaviloside B2). The isolated saponins showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines with the following rank: vaviloside B1/B2 > ascalonicoside A1/A2 > vaviloside A1/A2.     

The Bα and Bδ regulatory subunits of PP2A are necessary for assembly of the CaMKIV·PP2A signaling complex

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Bα or Bδ causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV·PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.     

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.