Adult-generated neurons exhibit diverse developmental fates

In mammals, olfactory bulb (OB) interneurons, granule cells and periglomerular (PG) cells, are generated throughout adulthood. PG cells comprise a heterogeneous population in both morphology and molecular phenotypes. It is unknown whether adult genesis of PG cells occurs among all subtypes or is limited to a subpopulation. We show that within 2 weeks after retroviral labeling in the subventricular zone, two morphological populations of PG cells are found in the OB, one with large multi-glomerular dendritic arbors, and one with dendritic arbors limited to one or two glomeruli. On both types, immature dendritic spines are first evident at 4 weeks and mature, pedunculated spines by 6 weeks. To differentiate PG subpopulations we used expression of calcium binding proteins, GAD67 and tyrosine hydroxylase as markers. Among adult-born BrdU labeled cells, all molecular subtypes were represented, although GAD67 and tyrosine hydroxylase expressing cells were overrepresented proportional to their expression in the total PG cell population. During the time when spines are maturing, approximately half the PG cells are lost, in roughly equal proportions to their generation. Our data show the diverse developmental potential of SVZ neuroblasts and suggest that integration into synaptic circuits is necessary for survival.     

Spatially distinct mitochondrial populations exhibit different mitofilin levels

Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria exhibit unique biochemical and functional properties; however, their association with structural membrane proteins that control mitochondrial morphology and functionality in striated muscle tissue was never reported. In IMF and SS mitochondria isolated from rat heart and gastrocnemius muscle, we analysed the expression levels of mitofilin, a mitochondria-associated protein involved in organelle structure maintenance. The statistically significant higher amounts of mitofilin detected in IMF compared with SS mitochondria, 37-fold in cardiac tissue and 3.8-fold in gastrocnemius, together with the specific energetic requirements of these mitochondrial populations highlight the importance of mitofilin in oxidative phosphorylation functionality and in mitochondrial plasticity in striated muscle. The differential expression levels of mitofilin between IMF and SS also suggest that this protein can be used as a specific molecular marker to comparatively discriminate spatially distant mitochondrial populations.     

不同日龄大鼠颈上交感神经节神经元电转染方法

目的 建立一种高效电转染不同日龄大鼠颈上交感神经节(superior cervical sympathetic ganglion,SCG)神经元细胞的方法.提高转染后细胞的成活率、转染效率和干扰效率.方法 用传统的及经改良的神经元培养液分别培养电转染后的7日龄、14日龄和40日龄SD大鼠SCG细胞,24 h后用台盼蓝染色方法观察并计算细胞成活率;通过改变质粒DNA和siRNA与转染液比例,优化转染条件,于转染24h后在共聚焦显微镜下观察并计算转染效率或干扰效率.结果 改良培养液可使14日龄以上SD大鼠SCG细胞转染后成活率达到75％以上,明显高于传统培养液转染后的成活率(P＜0.01),且结果稳定,细胞状态良好,能够满足后续实验研究的要求;优化转染条件后,DNA 的转染率及siRNA的干扰率显著提高,当DNA与转染液比例为1∶100(μg∶μL)时,细胞转染率最高;当siRNA与转染液比例为1∶50(μg∶ μL)时干扰率最高.结论 通过改良神经元培养液及优化转染条件,成功提高了电转染后细胞的成活率、转染效率和干扰效率,利用电转染方法可成功转染不同日龄SD大鼠SCG神经元.     

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.     

FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.     

Potential-dependent calcium channels in neurons from mollusks of varying ages

It was shown that with holding potentials (E_h) ranging from –50 to –30 mV the amplitudes of the incoming calcium current of identified neurons of the small parietal ganglion are significantly higher in elderly (20–24 month) Lymnaea stagnalis mollusks than in adult (10–12 months) ones.Gerontology Scientific-Research Institute, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 643–647, November–December, 1991.     

Isolated cell walls exhibit cation binding properties distinct from those of plant roots

Aims

The principal contributor to the cation binding properties of roots is currently considered to be the cell wall or, alternatively, the plasma membrane. The aim of this study was to highlight their respective contributions in the binding properties.

Methods

Cell walls of a dicotyledon (Solanum lycopersicum L.) and monocotyledon (Triticum aestivum L.) were isolated from roots and their binding properties were compared to those of their respective roots. Cell wall and root binding capacities were evaluated by potentiometric titrations and cation exchange capacity measurements, while their biochemical composition was analyzed by ¹³C-NMR spectroscopy.

Results

The lower binding capacity of isolated cell walls compared to roots revealed that cell plasma membranes had a higher binding site density than cell walls. The significant decrease in some NMR signals, i.e. carbonyl C, N alkyl/methoxyl C and alkyl C regions, suggested that carboxyl, amine and phosphate binding sites, borne by proteins and phospholipid plasma membranes, contribute to the binding capacity.

Conclusions

Cell walls and plasma membranes were found to be jointly involved in root binding properties and their respective contributions seemed vary between plants.     

白介素-6保护小脑颗粒神经元抗谷氨酸的神经毒性作用

目的：探讨白介素-6（IL-6）对谷氨酸诱导的神经元损伤的防治作用及其作用机制。方法：用IL-6慢性预处理培养的小脑颗粒神经元,然后后用谷氨酸急性刺激小脑颗粒神经元。用噻唑兰（MTT）比色法和末端脱氧核苷酸转移酶介导的原位缺口末端标记（TUNEL）法分别观察神经元的功能和凋亡的变化;用激光扫描共聚焦显微镜（LSCM）和逆转录聚合酶链式反应（RT—PCR）法分别检测神经元内Ca^2＋浓度的动态变化和IL-6信号转导蛋白gp130 mRNA的表达。结果：IL-6（2．5、5和10ng／ml）慢性预处理培养的小脑颗粒神经元,可浓度依赖性地改善谷氨酸诱导的神经元活性降低;并可明显减少谷氨酸诱导的神经元凋亡;还可显著抑制谷氨酸激发的神经元内Ca^2＋超载。此外。经IL-6慢性预处理的小脑颗粒神经元表达gp130mRNA明显低于未经IL-6预处理的神经元。结论：IL-6能保护神经元抵抗由谷氨酸诱导的兴奋毒性作用,IL-6的这种神经保护机制可能与它抑制神经元内Ca^2＋超载密切相关,而且可能由gp130细胞内信号转导途径介导。     

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Various forms of potentials recorded from caudate nucleus neurons

Spontaneous and evoked activity of caudate nucleus neurons was recorded extracellularly in acute experiments on cats. Different forms of potentials were found by analysis of the results. The potentials recorded belong to three types: ordinary action potentials; prepotentials or incomplete spikes differing from ordinary action potentials in their lower amplitude and slower decline, and complex discharges in which a spike of somewhat reduced amplitude is followed by a slow positive-negative wave. In the spontaneous activity prepotentials were observed both in complete action potentials and in isolation. The frequency of the complex discharges was 0.5–1 per second. The slow wave of these discharges blocked prepotential and action potential formation. The origin of these forms of potentials in neurons of the caudate nucleus is discussed and they are compared with analogous forms of potentials described for the Purkinje cells of the cerebellum.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukranian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 149–156, March–April, 1977.     

Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.     

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.     

Congeneric but still distinct: how closely related trypsin ligands exhibit different thermodynamic and structural properties

A congeneric series of benzamidine-type ligands with a central proline moiety and a terminal cycloalkyl group—linked by a secondary amine, ether, or methylene bridge—was synthesized as trypsin inhibitors. This series of inhibitors was investigated by isothermal titration calorimetry, crystal structure analysis in two crystal forms, and molecular dynamics simulations. Even though all of these congeneric ligands exhibited essentially the same affinity for trypsin, their binding profiles at the structural, dynamic, and thermodynamic levels are very distinct. The ligands display a pronounced enthalpy/entropy compensation that results in a nearly unchanged free energy of binding, even though individual enthalpy and entropy terms change significantly across the series. Crystal structures revealed that the secondary amine-linked analogs scatter over two distinct conformational families of binding modes that occupy either the inside or of the outside the protein's S3/S4 specificity pocket. In contrast, the ether-linked and methylene-linked ligands preferentially occupy the hydrophobic specificity pocket. This also explains why the latter ligands could only be crystallized in the conformationally restricting closed crystal form whereas the derivative with the highest residual mobility in the series escaped our attempts to crystallize it in the closed form; instead, a well-resolved structure could only be achieved in the open form with the ligand in disordered orientation. These distinct binding modes are supported by molecular dynamics simulations and correlate with the shifting enthalpic/entropic signatures of ligand binding. The examples demonstrate that, at the molecular level, binding modes and thermodynamic binding signatures can be very different even for closely related ligands. However, deviating binding profiles provide the opportunity to optimally address a given target.     

Structural characterization of glycosaminoglycans from zebrafish in different ages

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using ¹H-NMR.