Glycine-dependent activation of NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation requires two distinct agonists. Heterotetramers of two GluN1 and two GluN2 subunits, NMDA receptors are broadly distributed in the central nervous system, where they mediate excitatory currents in response to synaptic glutamate release. Pore opening depends on the concurrent presence of glycine, which modulates the amplitude and time course of the glutamate-elicited response. Gating schemes for fully glutamate- and glycine-bound NMDA receptors have been described in sufficient detail to bridge the gap between microscopic and macroscopic receptor behaviors; for several receptor isoforms, these schemes include glutamate-binding steps. We examined currents recorded from cell-attached patches containing one GluN1/GluN2A receptor in the presence of several glycine-site agonists and used kinetic modeling of these data to develop reaction schemes that include explicit glycine-binding steps. Based on the ability to match a series of experimentally observed macroscopic behaviors, we propose a model for activation of the glutamate-bound NMDA receptor by glycine that predicts apparent negative agonist cooperativity and glycine-dependent desensitization in the absence of changes in microscopic binding or desensitization rate constants. These results complete the basic steps of an NMDA receptor reaction scheme for the GluN1/GluN2A isoform and prompt a reevaluation of how glycine controls NMDA receptor activation. We anticipate that our model will provide a useful quantitative instrument to further probe mechanisms and structure–function relationships of NMDA receptors and to better understand the physiological and pathological implications of endogenous fluctuations in extracellular glycine concentrations.     

The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER in Caenorhabditis elegans

Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)–Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER. In a previous study, nematodes expressing a homologous mutation in the endogenous Caenorhabditis elegans serpin, srp-2, were reported to model the ER proteotoxicity induced by an allele of mutant neuroserpin. Our results suggest that SRP-2 lacks a classical N-terminal signal peptide and is a member of the intracellular serpin family. Using confocal imaging and an ER colocalization marker, we confirmed that GFP-tagged wild-type SRP-2 localized to the cytosol and not the ER. Similarly, the aggregation-prone SRP-2 mutant formed intracellular inclusions that localized to the cytosol. Interestingly, wild-type SRP-2, targeted to the ER by fusion to a cleavable N-terminal signal peptide, failed to be secreted and accumulated within the ER lumen. This ER retention phenotype is typical of other obligate intracellular serpins forced to translocate across the ER membrane. Neuroserpin is a secreted protein that inhibits trypsin-like proteinase. SRP-2 is a cytosolic serpin that inhibits lysosomal cysteine peptidases. We concluded that SRP-2 is neither an ortholog nor a functional homolog of neuroserpin. Furthermore, animals expressing an aggregation-prone mutation in SRP-2 do not model the ER proteotoxicity associated with FENIB.     

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

Background

Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.

Results

The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.

Conclusions

A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.     

Analysis of influenza virus hemagglutinin receptor binding mutants with limited receptor recognition properties and conditional replication characteristics

To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

Mitochondrial DNA variation in human metabolic rate and energy expenditure

The role of climate in driving selection of mtDNA as Homo sapiens migrated out of Africa into Eurasia remains controversial. We evaluated the role of mtDNA variation in resting metabolic rate (RMR) and total energy expenditure (TEE) among 294 older, community-dwelling African and European American adults from the Health, Aging and Body Composition Study. Common African haplogroups L0, L2 and L3 had significantly lower RMRs than European haplogroups H, JT and UK with haplogroup L1 RMR being intermediate to these groups. This study links mitochondrial haplogroups with ancestry-associated differences in metabolic rate and energy expenditure.     

Game-based biofeedback for paediatric anxiety and depression

Twenty-four children and adolescents aged 9-17 who were referred for treatment for anxiety were assigned to either a game-based biofeedback group or a waiting list comparison group. The eight-session biofeedback intervention included psychoeducation, identification of triggers and signs of anxiety, and in vivo practice. The intervention used computer-based gaming technology to teach and practise relaxation. Analyses using ANCOVA revealed significant differences in post-test scores of anxiety and depression measures between the two groups. The intervention group reduced anxiety and depression scores on standardised tests. Findings suggest that biofeedback-assisted relaxation training can be useful in decreasing anxiety and depressive symptoms in anxious youths.     

Visualizing clinical evidence: citation networks for the incubation periods of respiratory viral infections

Simply by repetition, medical facts can become enshrined as truth even when there is little empirical evidence supporting them. We present an intuitive and clear visual design for tracking the citation history of a particular scientific fact over time. We apply this method to data from a previously published literature review on the incubation period of nine respiratory viral infections. The resulting citation networks reveal that the conventional wisdom about the incubation period for these diseases was based on a small fraction of available data and in one case, on no retrievable empirical evidence. Overall, 50% of all incubation period statements did not provide a source for their estimate and 65% of original sources for incubation period data were not incorporated into subsequent publications. More standardized and widely available methods for visualizing these histories of medical evidence are needed to ensure that conventional wisdom cannot stray too far from empirically supported knowledge.     

Analysis of the microbial community and geochemistry of a sediment core from Great Slave Lake, Canada

Sediment cores taken from Great Slave Lake, Canada, were analysed to investigate their metabolically active microbial populations and geochemistry. The amplification of cDNA detected metabolically active bacterial (50 separate bands) and archaeal (49 separate band) communities. The bacterial communities were further resolved indicating active actinobacterial and γ-proteobacterial communities (36 and 43 individual bands respectively). Redundancy discriminate analysis and Monte Carlo permutation testing demonstrated the significant impact of geochemical parameters on microbial community structures. Geochemical analyses suggest that the upper 0.4 m represents soil weathering and erosion in the lake catchment. An increase in organic carbon in the lower core suggests either more primary productivity, indicating warmer climate conditions, associated with Holocene Climatic Optimum conditions pre 5,000 years BP or change from a reducing environment in the lower core to an oxidizing environment during more recent deposition. Drivers for bacterial, archaeal and actinobacterial community structures were sediment particle size, and its mineral composition. Depth also significantly affected γ- proteobacterial community structure. In contrast the organic carbon content did not significantly shape the microbial community structures within the sediment. This study indicates that geochemical parameters significantly contribute to microbial community structure in these sediments.