Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

金刷巴多糖的研究

金刷巴用热水提取,乙醇沉淀,精制得ＬＥＣ多糖,经Ｓｅｐｈａｄｅｘ Ｇ－１５０柱层析均为一性多糖。气相气谱检测含有葡萄糖,甘露糖,克分子比为Ｇｌｃ：Ｍａｎ＝２７：１,平均分子量为１１Ｘ１０＾５,糖含量为８２．４５％。经红外光谱分析,高碘酸氧化,Ｓｍｉｔｈ降解,ＬＥＣ多糖主要是β（１→３）甙键聚合成而的多支链多糖。     

Non‐motor parkinsonian pathology in aging A53T α‐Synuclein mice is associated with progressive synucleinopathy and altered enzymatic function

Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α–synuclein levels in substantia nigra pars compacta (SNc). Excess α‐synuclein spurs Lewy‐like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate‐limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α–synuclein for behavior and α–synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α‐synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α‐synuclein. Low adrenal PP2A activity co‐occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α–synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α‐synuclein pathology associated with pre‐motor stages of PD.

     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the “SOSIP” mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.     

A new yeast endosomal SNARE related to mammalian syntaxin 8

We report the identification of a yeast SNARE that has escaped notice because of an apparent error in the genome sequence and because it is functionally redundant. It is encoded by an extended version of ORF YAL014c, and since its SNARE motif is related to mammalian syntaxin 8 we term the gene SYN8 . Syn8p is in endosomes. Co-precipitation indicates a set of complexes containing Pep12p, Vti1p, either Syn8p or Tlg1p and either Snc1p or Ykt6p. Analysis of growth and trafficking defects demonstrates that in the absence of Tlg1p, Syn8p is required for Pep12p function. Conversely, when Tlg1p is present, Syn8p can be removed without loss of Pep12p function, or induction of any other obvious trafficking defect. Syn8p thus appears to be a functional homolog of mammalian syntaxin 8, but Tlg1p can, amongst other roles, provide an equivalent function .     

Metal-stabilized rare tautomers of nucleobases

 A Pt^II complex containing N4 bound neutral 1-methylcytosine (1-MeC), trans–[Pt(NH₃)₂(1-MeC-N4)₂](ClO₄)₂ (5), has been prepared and characterized by X-ray analysis. The complex contains the rare iminooxo tautomer form of the cytosine nucleobase. Pt^II binding is through the exocyclic N4 position of the nucleobases, with Pt and the N3 positions in a syn orientation. As a consequence, the proton at N3 is pointing toward the heavy metal, thereby allowing an agostic Pt···HN interaction. Formation of 5 is achieved via oxidation of the linkage isomer trans–[Pt(NH₃)₂(1-MeC-N3)₂]²⁺ (1) to a Pt^IV species (2), followed by metal migration to N4, and subsequent reduction to Pt^II. This process is a text-book example for a redox-assisted metal migration at a heterocyclic ligand. The existence of various rotamers of 5 in aqueous solution is evident from ¹H NMR spectroscopy. The possible role of these rotamers of the metalated rare tautomer, and in particular of those having Pt and the N3 position in an anti arrangement, with regard to base mispairing is discussed. Received: 27 February 1996 / Accepted: 10 June 1996     

Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [³H]ERGO in several brain regions of octn1^−/− mice was much lower than that in wild-type mice, whereas extracellular marker [¹⁴C]mannitol exhibited similar distribution in the two strains. The [³H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [³H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [³H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development.     

Crystal and molecular structures of syn and anti isomers of [MoO(μ-NAr)(S2CNR2)]2 (R = Et, Pr; Ar = Ph, p-tolyl)

Crystallographic studies have been carried out on four complexes of the type, [MoO(μ-NAr)(S₂CNR₂)]₂ (R = Et, Pr; Ar = Ph, p-tol). Each contains a binuclear molybdenum(V) centre bridged by two imido ligands, with each molybdenum atom also carrying terminal dithiocarbamate and oxo moieties. Syn and anti structural types are seen which result in significant differences within the Mo₂N₂ core; that in the anti structures being planar, while fold angles of 151.1° and 164.6° are observed in the syn complexes.     

The synapsins: multitask modulators of neuronal development

Neurons are examples of specialized cells that evolved the extraordinary ability to transmit electrochemical information in complex networks of interconnected cells. During their development, neurons undergo precisely regulated processes that define their lineage, positioning, morphogenesis and pattern of activity. The events leading to the establishment of functional neuronal networks follow a number of key steps, including asymmetric cell division from neuronal precursors, migration, establishment of polarity, neurite outgrowth and synaptogenesis. Synapsins are a family of abundant neuronal phosphoproteins that have been extensively studied for their role in the regulation of neurotransmission in presynaptic terminals. Beside their implication in the homeostasis of adult cells, synapsins influence the development of young neurons, interacting with cytoskeletal and vesicular components and regulating their dynamics. Although the exact molecular mechanisms determining synapsin function in neuronal development are still largely unknown, in this review we summarize the most important literature on the subject, providing a conceptual framework for the progress of present and future research.