Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus

Hippocampal injury-associated learning and memory deficits are frequent hallmarks of brain trauma and are the most enduring and devastating consequences following traumatic brain injury (TBI). Several reports, including our recent paper, showed that TBI brought on by a moderate level of controlled cortical impact (CCI) induces immature newborn neuron death in the hippocampal dentate gyrus. In contrast, the majority of mature neurons are spared. Less research has been focused on these spared neurons, which may also be injured or compromised by TBI. Here we examined the dendrite morphologies, dendritic spines, and synaptic structures using a genetic approach in combination with immunohistochemistry and Golgi staining. We found that although most of the mature granular neurons were spared following TBI at a moderate level of impact, they exhibited dramatic dendritic beading and fragmentation, decreased number of dendritic branches, and a lower density of dendritic spines, particularly the mushroom-shaped mature spines. Further studies showed that the density of synapses in the molecular layer of the hippocampal dentate gyrus was significantly reduced. The electrophysiological activity of neurons was impaired as well. These results indicate that TBI not only induces cell death in immature granular neurons, it also causes significant dendritic and synaptic degeneration in pathohistology. TBI also impairs the function of the spared mature granular neurons in the hippocampal dentate gyrus. These observations point to a potential anatomic substrate to explain, in part, the development of posttraumatic memory deficits. They also indicate that dendritic damage in the hippocampal dentate gyrus may serve as a therapeutic target following TBI.     

密粘褶菌胞外低分子量多肽在纤维素降解中作用的研究

从褐腐真菌中能强烈降解纤维素的代表菌株密粘褶菌(Gloeophyllum trabeum)的胞外酶液中首次分离纯化得到一低分子量的活性多肽组分(称作Gt因子),此组分能在有O.2和Fe³⁺存在时产生羟基自由基HO·;对纤维素降解的研究表明,Gt因子不同于纤维素酶对纤维素的β1.4糖苷键的水解作用,而以HO·氧化的途径作用于纤维素,导致纤维素中氢键的断裂,降低纤维素的结晶度,使其暴露出更多的末端,从而有利于纤维素的进一步降解。     

Delimiting the coastal geographic background to predict potential distribution of Spartina alterniflora

We examined crustacean zooplankton data (excluding nauplii) from 15 shallow lakes in south and central Florida, spanning a range of sizes and Chlorophyll-a concentrations. Each dataset was comprised of monthly samples from 2 years (6 lakes), or monthly to quarterly samples from 10 or 12 years (9 lakes). We quantified relationships between the zooplankton to phytoplankton biomass ratio (BZ:BP) and measurements of BZ and BP at three levels of resolution: (1) sampling events; (2) seasonal means; and (3) period-of-record means and medians. For individual sampling events, variations in ZB explained most of the variation in BZ:BP and ratios were little affected by changes in BP. Seasonal declines in BZ:BP corresponded with declines in BZ, were not related to declines in biomass of edible algae, and happened in spring–summer when earlier studies indicated high densities of planktivorous fish. Period-of-record means and medians did not identify any relationships between the biomass ratio and either BZ or BP, suggesting that processes affecting the ratio operate at shorter time scales than multiple years. Short-term and seasonal changes in BZ:BP in Florida may be controlled by predation. Testing this hypothesis will require coincident sampling of plankton and fish over a number of years or experimental studies.     

Enhancement of the direct antimicrobial activity of Lysep3 against <Emphasis Type="Italic">Escherichia coli</Emphasis> by inserting cationic peptides into its C terminus

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.     

Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用

我们已证实周期蛋白激酶（cyclin-dependent kinases）cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡。本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用。结果显示,roscovitine（50μmol／L）处理PC12细胞12h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42％。非特异性caspases抑制剂Z-VAD-FMK（50μmol／L）和caspase-3特异性抑制剂Z-DEVD-FMK（100μmol／L）可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29．03％（roscovitine）增至58．06％（Z-VAD-FMK＋roscovitine）和45．16％（Z-DEVD-FMK＋roscovitine）：用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectfin 120kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases参与roscovitine所敛的细胞凋亡,其中caspase-3发挥重要作用。     

毕赤酵母表达体系中重组蛋白的分离纯化

随着基因重组技术的快速发展,基因工程产品的利用越来越广泛,但其分离纯化的成本约占总成本的60%～70%.因此,探索一些简单有效的分离纯化方法尤为必要.简单介绍了目前较为流行的毕赤酵母表达体系,着重概述了重组蛋白分离纯化技术方法的应用情况.     

Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2: CLEAVING NOT ONLY GLYCYL BONDS IN THE HYDROPHOBIC REGIONS BUT ALSO PEPTIDE BONDS IN THE HYDROPHILIC REGIONS INVOLVED IN CROSS-LINKING*

Elastin is a common insoluble protein that is abundant in marine vertebrates, and for this reason its degradation is important for the recycling of marine nitrogen. It is still unclear how marine elastin is degraded because of the limited study of marine elastases. Here, a novel protease belonging to the M23A subfamily, secreted by Pseudoalteromonas sp. CF6-2 from deep-sea sediment, was purified and characterized, and its elastolytic mechanism was studied. This protease, named pseudoalterin, has low identities (<40%) to the known M23 proteases. Pseudoalterin has a narrow specificity but high activity toward elastin. Analysis of the cleavage sites of pseudoalterin on elastin showed that pseudoalterin cleaves the glycyl bonds in hydrophobic regions and the peptide bonds Ala–Ala, Ala–Lys, and Lys–Ala involved in cross-linking. Two peptic derivatives of desmosine, desmosine-Ala-Ala and desmosine-Ala-Ala-Ala, were detected in the elastin hydrolysate, indicating that pseudoalterin can dissociate cross-linked elastin. These results reveal a new elastolytic mechanism of the M23 protease pseudoalterin, which is different from the reported mechanism where the M23 proteases only cleave glycyl bonds in elastin. Genome analysis suggests that M23 proteases may be popular in deep-sea sediments, implying their important role in elastin degradation. An elastin degradation model of pseudoalterin was proposed, based on these results and scanning electron microscopic analysis of the degradation by pseudoalterin of bovine elastin and cross-linked recombinant tropoelastin. Our results shed light on the mechanism of elastin degradation in deep-sea sediment.     

Human amniotic fluid-derived c-kit(+) and c-kit (-) stem cells: growth characteristics and some differentiation potential capacities comparison

Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit⁺ stem cells (c-kit⁺ AFS) were compared with the c-kit⁻ (unselected) stem cells (c-kit⁻ AFS). Our findings revealed that the optimal period to obtain c-kit⁺ AFS cells was between 16 and 22 weeks of gestation. Following cell sorting, c-kit⁺ AFS cells shared similar morphological and proliferative characteristics as the c-kit⁻ AFS cells. Both c-kit⁺ and c-kit⁻ AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit⁺ and c-kit⁻ AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit⁺ AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit⁻ AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit⁺ AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy.     

Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus

ABSTRACT: BACKGROUND: The incidence of PHE among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. Therefore, the development of a sensitive, specific, and easily-performed assay is crucial for the rapid detection and surveillance of PHE-CoV infection and transmission. RESULTS: An immunochromatographic strip was developed for the detection of PHE-CoV. The colloidal gold-labeled MAb 4D4 was used as the detection reagent, and the MAb 1E2 and goat anti-mouse IgG coated the strip's test and control lines, respectively. The immunochromatographic strip was capable of specifically detecting PHE-CoV with a HA unit of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4 [DEGREE SIGN]C for 12 months did not change their sensitivity or specificity. Using RT-PCR as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 100% and 97.78%, respectively. There was an excellent agreement between the results obtained by RT-PCR and the immunochromatographic strips (kappa = 0.976). Additionally, there was a strong agreement between the sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatographic strips (Kappa = 0.976). When the immunochromatographic strips were used for diagnosing PHE-CoV infection in the Jilin Province, the PHE-CoV-positive rate ranged from 61.54% in the Jilin district to 17.95% in the Songyuan district. CONCLUSIONS: Based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of PHE-CoV for surveillance and epidemiological purposes.