Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

The PIP5K2A and RGS4 genes are differentially associated with deficit and non-deficit schizophrenia

Several putative schizophrenia susceptibility genes have recently been reported, but it is not clear whether these genes are associated with schizophrenia in general or with specific disease subtypes. In a previous study, we found an association of the neuregulin 1 (NRG1) gene with non-deficit schizophrenia only. We now report an association study of four schizophrenia candidate genes in patients with and without deficit schizophrenia, which is characterized by severe and enduring negative symptoms. Single-nucleotide polymorphisms (SNPs) were genotyped in the DTNBP1 (dysbindin), G72/G30 and RGS4 genes, and the relatively unknown PIP5K2A gene, which is located in a region of linkage with both schizophrenia and bipolar disorder. The sample consisted of 273 Dutch schizophrenia patients, 146 of whom were diagnosed with deficit schizophrenia and 580 controls. The strongest evidence for association was found for the A-allele of SNP rs10828317 in the PIP5K2A gene, which was associated with both clinical subtypes (P = 0.0004 in the entire group; non-deficit P = 0.016, deficit P = 0.002). Interestingly, this SNP leads to a change in protein composition. In RGS4, the G-allele of the previously reported SNP RGS4-1 (single and as part of haplotypes with SNP RGS4-18) was associated with non-deficit schizophrenia (P = 0.03) but not with deficit schizophrenia (P = 0.79). SNPs in the DTNBP1 and G72/G30 genes were not significantly associated in any group. In conclusion, our data provide further evidence that specific genes may be involved in different schizophrenia subtypes and suggest that the PIP5K2A gene deserves further study as a general susceptibility gene for schizophrenia.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

ACE I/D and MTHFR C677T polymorphisms are significantly associated with type 2 diabetes in Arab ethnicity: A meta-analysis

In this meta-analysis study, SNPs were investigated for their association with type 2 diabetes (T2D) in both Arab and Caucasian ethnicities. A total of 55 SNPs were analyzed, of which 11 fulfilled the selection criteria, and were used for analysis. It was found that TCF7L2 rs7903146 was significantly associated with a pooled OR of 1.155 (95%C.I. = 1.059–1.259), p < 0.0001 and I² = 78.30% among the Arab population, whereas among Caucasians, the pooled OR was 1.45 (95%C.I. = 1.386–1.516), p < 0.0001 and I² = 77.20%. KCNJ11 rs5219 was significantly associated in both the populations with a pooled OR of 1.176(1.092–1.268), p < 0.0001 and I² = 32.40% in Caucasians and a pooled OR of 1.28(1.111–1.475), p = 0.001 among Arabs. The ACE I/D polymorphism was found to be significantly associated with a pooled OR of 1.992 (95%C.I. = 1.774–2.236), p < 0.0001 and I² = 83.20% among the Arab population, whereas among Caucasians, the pooled OR was 1.078 (95%C.I. = 0.993–1.17), p = 0.073 and I² = 0%. Similarly, MTHFR C677T polymorphism was also found to be significantly associated among Arabs with a pooled OR of 1.924 (95%C.I. = 1.606–2.304), p < 0.0001 and I² = 27.20%, whereas among Caucasians, the pooled OR was 0.986 (95%C.I. = 0.868–1.122), p = 0.835 and I² = 0%. Meanwhile PPARG-2 Pro12Ala, CDKN2A/2B rs10811661, IGF2BP2 rs4402960, HHEX rs7923837, CDKAL1 rs7754840, EXT2 rs1113132 and SLC30A8 rs13266634 were found to have no significant association with T2D among Arabs. In conclusion, it seems from this study that both Arabs and Caucasians have different SNPs associated with T2D. Moreover, this study sheds light on the profound necessity for further investigations addressing the question of the genetic components of T2D in Arabs.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli

Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation.     

Effect of selenium-enriched malt on hepatocarcinogenesis, paraneoplastic syndrome and the hormones regulating blood glucose in rats treated by diethylnitrosamine

233 SD rats weighing 100 approximately 120 g were divided randomly into 6 groups. The animals in group I and group II received 0.1 mg/kg selenium in the form of sodium selenite only and served as the negative control and positive control, respectively. Animals in groups III, IV and V were fed with selenium as Se-enriched malt supplemented diets (0.3, 1 and 3 mg/kg), and group VI with selenium by using sodium selenite supplemented diets (3 mg/kg). Animals of groups II approximately VI were induced hepatoma by diethylnitrosamine (100 mg/l) for 16 weeks, then drunk with sterilized water for 2 more weeks. Subsequently, the effects of Se-enriched malt and sodium selenite on hepatoma nodules, relative liver weight, the liver function indices including alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), total bilirubin (TBIL), and the tumor markers, named as gamma-glutamyltranspeptidase (GGT), alpha-fetoprotein (AFP), insulin-like growth factor-II (IGF-II) were recorded. The calcium concentration, glucose content in plasma and values of the hormones regulating blood glucose, such as insulin, glucagons and thyroid hormones (3,5,3'-tetraiodothyronine, T(3); 3,5,3'5'-tetraiodothyronine, T(4)) were observed as well. At the same time, the correlations between the concentration of plasma glucose and related hormones were also analyzed. The results indicated that Se-enriched malt showed a better chemopreventive efficiency in decreasing the number of hepatoma nodules, relative liver weight and the contents of AFP, GGT, IGF-II, ALT, ALP and TBIL in the plasma, and delaying the descent of hormones in the serum, names as insulin, glucagons, T(3) and T(4) than those feeding with sodium selenite. Effect of Se-enriched malt excelled sodium selenite in the aspects of deadening the descent of glucose concentration in the plasma and the rise of calcium concentration in the serum of the rats with hepatoma induced by diethylnitrosamine. The values of glucose and calcium were significantly related to those items fore-named. In conclusion, the function of Se-enriched malt in deadening the lesion and delaying the development of hepatoma of rats induced by diethylnitrosamine was better than that of sodium selenite. Hypoglycemia and hypercalcemia were significantly correlated with the multifactors mentioned above.