Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds

Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained from other sterilization methods. The results of the maximum stress test and Young’s modulus showed that gamma-irradiated and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released, which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering.     

Multi-Stability and Multi-Instability Phenomena in a Mathematical Model of Tumor-Immune-Virus Interactions

Recent advances in virology, gene therapy, and molecular and cell biology have provided insight into the mechanisms through which viruses can boost the anti-tumor immune response, or can infect and directly kill tumor cells. A recent experimental report (Bridle et al. in Molec. Ther. 18(8):1430–1439, 2010) showed that a sequential treatment approach that involves two viruses that carry the same tumor antigen leads to an improved anti-tumor response compared to the effect of each virus alone. In this article, we derive a mathematical model to investigate the anti-tumor effect of two viruses, and their interactions with the immune cells. We discuss the conditions necessary for permanent tumor elimination and, in this context, we stress the importance of investigating the long-term effect of non-linear interactions. In particular, we discuss multi-stability and multi-instability, two complex phenomena that can cause abrupt transitions between different states in biological and physical systems. In the context of cancer immunotherapies, the transitions between a tumor-free and a tumor-present state have so far been associated with the multi-stability phenomenon. Here, we show that multi-instability can also cause the system to switch from one state to the other. In addition, we show that the multi-stability is driven by the immune response, while the multi-instability is driven by the presence of the virus.     

Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

Nucleotide Binding Domain Interactions During the Mechanochemical Reaction Cycle of ATP-Binding Cassette Transporters

ATP-binding cassette (ABC) transporters serve as importers and exporters for a wide variety of solutes in both prokaryotes and eukaryotes, and are implicated in microbial drug resistance and a number of significant human genetic disorders. Initial crystal structures of the soluble nucleotide binding domains (NBDs) of ABC transporters, while a significant step towards understanding the coupling of ATP binding and hydrolysis to transport, presented researchers with important questions surrounding the role of the signature sequence residues, the composition of the nucleotide binding sites, and the mode of NBD dimerization during the transport reaction cycle. Recent studies have begun to address these concerns. This mini-review summarizes the biochemical and structural characterizations of two archaebacterial NBDs from Methanocaldococcus jannaschii, MJ0796 and MJ1267, and offers current perspectives on the functional mechanism of ABC transporters.     

从一名国内感染的艾滋病人分离人免疫缺陷病毒（HIV）

从一名国内感染的艾滋病人采血,分离其外周血单核细胞（ＰＭＣｓ）。首先与正常的ＰＭＣｓ共培养,４周后检测其ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ）达到峰值。用此时的细胞及其上清分别感染Ｊｕｒｋａｔ－ｔａｔ、ＣＥＭ、ＭＴ４细胞,可很快地在这三株细胞中检测到ＨＩＶ生长,ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中生长情况最好,同时用病人血清直接感染Ｊｕｒｋａｔ－ｔａｔ和ＭＴ４细胞,４周后检测其细胞上清ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ法）为阳性,但ＯＤ值很低（约为ＰＭＣ共培养组的一半）。用病人的少量全血与正常ＰＭＣｓ共培养,得到的结果与分离病人ＰＭＣｓ法相近。应用间接免疫荧光法（ＩＦＡ）、免疫酶法（ＩＥＡ）、蛋白印迹法及ＨＩＶ－１ＰＯｌ基因和Ｅｎｖ基因特异引物的聚合酶链反应（ＰＣＲ）等证实为ＨＩＶ－１病毒。分离的ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中连续传代,细胞被感染后２－３天即出现以大量融合细胞为主的细胞病变,感染后７－１０天细胞几乎全部死亡。病毒在连续传代过程中的生长特征及致细胞病变特征不变。此病毒命名为ＣＡ－２毒株。     

不同采收期地肤子中皂甙含量的变化

地肤子为藜科植物地肤〔Ｋｏｃｈｉａｓｃｏｐａｒｉａ (Ｌ .)Ｓｃｈｒａｄ .〕的干燥成熟果实 ,始载于《神农本草经》 ,具有“治膀胱热、利小便、益精气”等功效 ,久服能“耳聪目明、轻身、耐老”。药理研究表明地肤子中所含的三萜皂甙类成分为其主要活性成分 ,具有抗炎、抗过敏和抗搔痒等作用[1 ,2 ] 。地肤子为一年生草本 ,一般于 4月上、中旬栽种 ,花期为 7- 9月 ,果期为 8- 10月[3] ,传统经验是在秋季果实成熟时采收 ,但何时采收其有效成分含量最高 ,这方面尚未见研究和报道。因此 ,本文分别采用高效液相色谱法和比色法检测不同采收期…     

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (−) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.     

Biochemical synthesis of novel,self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Purpose of work  

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds.