The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.     

TyrB26位点改变的人类胰岛素类似物的化学合成与体外活性

摘要：为了研究人类胰岛素B链第26位的酪氨酸对胰岛素和受体之间的结合的影响,包括单独的氨基酸替换或化合物替换的不同的胰岛素类似物被合成,其中化合物替代的类似物的B链C末端都减少了4个氨基酸。在对它们与胰岛素受体的亲和力进行研究中,结果发现它们与胰岛素受体的亲和力没有丢失, HisB26类似物和N-MeHisB26类似物的结合能力与胰岛素相比改变不大,分别是胰岛素的72 %和107 %。N-MeGluB26类似物,AadB26类似物和Phe (4-carboxy) B26类似物的结合能力有很大的提高,分别是130 %, 234 %和160 %。     

Kinetic analysis of the transformation of phthalate esters in a series of stoichiometric reactions in anaerobic wastes

Phthalates such as dimethyl phthalate, dimethyl terephthalate (DMT), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate (MEHP) are degraded to varying degrees under anaerobic conditions in waste treatment systems. Here we kinetically analyse the enzymatic hydrolyses involved and the subsequent stoichiometric reactions. The resulting model indicates that the degradation of the alcohols released and the transformation of the phthalic acid (PA) result in biphasic kinetics for the methane formation during transformation of DMT, DEP and MEHP. The ester hydrolysis and the PA transformation to methane appear to be the two rate-limiting steps. The PA-fermenting bacteria, which have biomass-specific growth rates between 0.04 and 0.085 day⁻¹, grow more slowly than the other bacteria involved. Anaerobic microorganisms that remove intermediate products during phthalic acid ester conversion appear to be important for the efficiency of the ultimate phthalate degradation and to be inhibited by elevated hydrogen partial pressures. The model was based on (and the simulations corresponded well with) data obtained from experimental waste treatment systems.     

Hair Trace Elements in Overweight and Obese Adults in Association with Metabolic Parameters

The objective of the present study was to investigate the level of toxic and essential trace elements in hair of adult overweight and obese persons as well as its association with metabolic parameters. Hair trace element levels were assessed using inductively coupled plasma mass-spectrometry in 112 overweight and obese patients and 106 lean controls. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose, uric acid (UA) levels, and cholinesterase (CE) and gamma-glutamyltransferase (GGT) activity were also assessed. Excessive body weight significantly affected hair trace element levels. In particular, hair Co (33%), Cu (13%), I (30%), Mg (2-fold), Mn (25%), Zn (17%), and Ni (21%) levels were lower, whereas Al (14%) and As levels were higher in comparison to those in the control group. Correlation analysis demonstrated the most significant correlations for hair Mg with body weight, BMI, systolic and diastolic blood pressure, and UA, and for hair Al with body weight, BMI, TC, glucose, TG, CE, GGT, and UA. Multiple regression analysis demonstrated that trace elements were not associated with TC and LDL-C levels neither in crude nor in adjusted models. In turn, crude and adjusted models accounted for 25 and 43% of serum TG variance. The most significant associations were observed for hair Al, Fe, Si, and V in adjusted model. The obtained data demonstrate that obesity-related metabolic disorders may be at least partially mediated by altered trace element and mineral levels.     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996