Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

Effects of 3-Methylgibberellin Analogs on Gibberellin 3β-Hydroxylases and Plant Growth

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉· inhibited the conversion of [2, 3-³H2]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P. vulgaris and C. maxima at 3 μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.     

Action of ω-conotoxin on calcium currents of rat pituitary GH3 cells

The effect of -conotoxin (-CgTX) on calcium currents of rat pituitary GH3 cells was studied by the voltage clamp method on a whole cell, under tight junction conditions. Two different components of the inward calcium current were observed in a solution containing 15 mM Ca²⁺. The first was activated with a holding potential of –80 mV and by testing pulses more positive than –55 mV. A shift of holding potential to –40 mV led to steady-state inactivation of this low-threshold component of the current. -CgTX at the initial moment after its application had an activating action on both components of the calcium current: low-threshold and high-threshold, but the increase in the first was much greater. In the present experiments the currents increased as early as 30 sec after replacement of the external solution; later the drop of current took place with temporal parameters characteristic of the spontaneous current drop in the control solution during cell dialysis. Incubation of the cells in growth medium containing 5 µM -CgTX for 2 h led to an increase in the density of both types of calcium currents in the GH3 cells, which was reduced after incubation for 2 h in the same medium. Thus -CgTX was found to have an activating action on calcium currents of GH3 cells at the initial moment after application of the toxin. The absence of a marked blocking action of -CgTX on the calcium currents of the test cells confirms the high tissue specificity of action of the toxin as a blocker of high-threshold calcium channels in the nerve cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 199–205, March–April, 1991.     

Impact of Bacterial NO3? Transport on Sediment Biogeochemistry

Experiments demonstrated that Beggiatoa could induce a H₂S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO₃⁻ reduction from denitrification to dissimilatory NO₃⁻ reduction to ammonium. pH, O₂, and H₂S profiles indicated that the bacteria oxidized H₂S with NO₃⁻ and transported S⁰ to the sediment surface for aerobic oxidation.     

Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a–e, 4a–i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f–h), could have anticancer properties.     

Stability of plant mRNAs depends on the length of the 3′-untranslated region

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.     

Effects of DNA3′pp5′G capping on 3′ end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance

When DNA breakage results in a 3′-PO₄ terminus, the end is considered ‘dirty’ because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3′-PO₄ to form a DNA_3′pp_5′G_OH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD⁺-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3′-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.     

Effects of methylation of deiodinase 3 gene on gene expression and severity of Kashin–Beck disease

Kashin–Beck disease (KBD) is a complex endemic osteoarthropathy, which mainly occurs in the northeast to southwest China. Iodothyronine deiodinases 3 (DIO3) is one of the selenoproteins, which is closely related to bone metabolism and unclear to KBD. This study aims to investigate the role and associated mechanisms of methylation and expression of DIO3 with disease severity in patients with KBD. We performed a bioinformatics analysis first to identify the biological mechanisms involved in selenoproteins. The methylation status of the DIO3 gene and DIO3 gene expression, as well as DIO3-related regulatory genes in patients with KBD, were analyzed. We found that 15 CpG sites of six selenoproteins were hypomethylated with 5-azacytidine treatment. DIO3 hypermethylation was associated with an increased risk of KBD and may lead to downregulation of DIO3 gene expression as well as be an indicator of the severity of KBD, which may provide a new insight for gene–environment correlations and interactions in etiology and pathogenesis of KBD.     

Free energy calculations on the stability of the 14-3-3ζ protein

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.     

The Impact of 3′UTR Variants on Differential Expression of Candidate Cancer Susceptibility Genes

Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. Skin tumor susceptibility 5 (Skts5) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3′ untranslated region (3′UTR) of genes at Skts5 influenced expression, we conducted a systematic evaluation of 3′UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3′UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3′UTRs of six genes, Bcap29, Dgkb, Hbp1, Pik3cg, Twistnb, and Tspan13 differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3′UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. Ifrd1 and Etv1 showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3′UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.     

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer's disease (AD). Recent studies of the physiological correlation between tau and α-synuclein (α-SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α-SN; and (b) the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α-SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α-SN-mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α-SN directly stimulates the phosphorylation of tau by glycogen synthase kinase-3β (GSK-3β), (b) α-SN forms a heterotrimeric complex with tau and GSK-3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α-SN are responsible for the stimulation of GSK-3β-mediated tau phosphorylation. Thus, it is concluded that α-SN functions as a connecting mediator for tau and GSK-3β, resulting in GSK-3β-mediated tau phosphorylation. Because the expression of α-SN is promoted by oxidative stress, the accumulation of α-SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK-3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α-SN-induced phosphorylation of tau by GSK-3β through its direct binding to α-SN, suggesting that Hsp70 acts as a physiological suppressor of α-SN-mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α-SN-mediated tau phosphorylation in the initial stage of neurodegenerative disease.     

Effects of cell surface α2-3 sialic acid on osteogenesis

A cell culture model of osteoblast differentiation was applied in our study of the effect of sialic acid on the osteogenesis by using the pre-osteoblast of MC3T3-E1 subclone 14 cells. Following the treatment of different concentrations of α2,3-neuraminidase, which specifically removed the α2-3 sialic acid from cell surface, a significant decrease of α2-3 sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-II) by flow cytometry analysis. von Kossa staining showed that the bone mineralization decreased in MC3T3-E1 subclone 14 cells after the treatment of α2,3-neuraminidase for 2 weeks. However α2,3-neuraminidase did not affect the formation of osteoblasts in MC3T3-E1 subclone 14 cells, which was demonstrated by positive alkaline phosphatase (ALP)-staining. Characteristic biological markers and osteoblast-like cell-related factors of osteoblastic cells were also examined. Both RT-PCR and Western blot analysis demonstrated that the expression of bone sialoprotein (BSP), osteoprotegerin (OPG), and vitamin D receptor (VDR) were significantly decreased when α2-3 sialic acid expression decreased on the cell surface, while the expression of osteocalcin (OC) and osteopontin (OPN) remained unchanged. We propose a hypothesis that α2-3 sialic acid affects bone mineralization but not osteogenic differentiation.     

Evidence of positive selection on the Atlantic salmon CD3γδ gene

Atlantic salmon are typically anadromous, spending the majority of their lifetime in oceans and returning to fresh water to breed. This diversity of environments likely results in strong selective forces shaping their genome. In this paper, we present the first genomics approach to detect positive selection operating on the Salmo salar (salmon) lineage, an important aquaculture species. We identify a panel of candidate genes that may have been subject to adaptive evolution in this species. In particular, we identify a robust signature of positive selection operating on the salmon CD3γδ gene, which encodes one of the protein chains essential for formation of the T-cell receptor complex and for T-cell activation. Furthermore, we identified the particular codon sites that have been subject to positive selection in fish and highlight two sites flanking an important N-glycosylation site in this molecule. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Identification of an autosomal recessive stuttering locus on chromosome 3q13.2–3q13.33

Stuttering is a common speech disorder with substantial genetic contributions. To better understand the genetic factors involved in stuttering, we performed a genome-wide linkage study in a newly-ascertained consanguineous stuttering family from Pakistan. A linkage scan in this family using parametric linkage analysis revealed significant linkage only on chromosome 3q13.2–3q13.33, with a maximum two-point LOD score of 4.23 under an autosomal recessive model of inheritance.