Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.     

A novel algesic peptide derived from skin secretions of the frog Amolops loloensis.

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL and isolated from skin secretion of the frog Amolops loloensis, is shown to strengthen nociceptive responses induced by inflammatory factors and strongly inhibit the contraction of isolated ileum. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. These two types of responses seem to be a part of the defensive functions against predators or aggressors. The current results suggest that pharmacological molecules in amphibian skins not only act as innate defense mechanisms against microorganisms but also exert other defensive physiological functions against other aggressors.     

Tumour necrosis factor-alpha receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage

OBJECTIVES: The study investigated the association of TNFR1 gene polymorphism with early recurrent spontaneous miscarriage (ERSM) in Chinese women, and soluble TNFR1 (sTNFR1) expression in ERSM women. STUDY DESIGN: Two single nucleotide polymorphisms (SNPs) located at -383 (AGA to AGC) in the promoter region and +36 (CCA to CCG) in exon 1 of TNFR1 were investigated in 188 non-pregnant ERSM Chinese women. The serum sTNFR1 was measured by the ELISA method. RESULTS: Both SNPs were not associated with ERSM. The non-pregnant ERSM women had significantly higher levels of serum sTNFR1, compared with the non-pregnant, normal women (1.84+/-0.54 ng/ml versus 1.62+/-0.38 ng/ml; t=-2.053; p<0.05). CONCLUSIONS: The data do not provide evidence that TNFR1 gene polymorphism is etiologically important for ERSM in Chinese women. But, a significantly raised sTNFR1 level in non-pregnant ERSM women was recorded compared to women with normal pregnancies. The result suggests that pregnancy failure is associated with an increase of sTNFR1.     

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.     

Structured to reduce the mitogenicity of anti-CD3 antibody based on computer-guided molecular design

The mouse anti-human CD3 monoclonal antibody such as OKT3 is a potent immunosuppressive agent used in clinical transplantation to manipulate T-cell functions and prevent acute allograft rejection. However, the broad use of anti-CD3 antibody in clinical treatment was severely limited by the side effects of human anti-mouse antibody response and cytokine release syndrome. In this study, on the basis of a murine anti-human CD3 antibody yCD3 obtained in our previous work, a novel engineered anti-human CD3 antibody fragment (i.e. V(H)-Linker-V(L)-Hinge-CH(3)) was constructed with computer-guided molecular design method to avoid the clinical side effects. According to the distance geometry and intra-molecular interaction, the hinge region was re-designed and different from the parental hinge region in human IgG1. With the novel hinge region, the cysteine residues in hinge were exposure and prone to form the disulfide bond. Therefore, a novel bivalent antibody fragment named as mini-yCD3 was obtained. Mini-yCD3 displayed similar antigen-binding affinity and specificity to yCD3. Importantly, mini-yCD3 was shown to be much less potent in the induction of T-cell proliferation, cytokine release (interferon-gamma and interleukin-2) and early activation marker expression on the cell surface (CD69 and CD25) than parental yCD3. Furthermore, mini-yCD3 was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with similarity as yCD3. In conclusion, the constructed mini-yCD3 was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to yCD3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.     

Assembling millions of short DNA sequences using SSAKE

Novel DNA sequencing technologies with the potential for up to three orders magnitude more sequence throughput than conventional Sanger sequencing are emerging. The instrument now available from Solexa Ltd, produces millions of short DNA sequences of 25 nt each. Due to ubiquitous repeats in large genomes and the inability of short sequences to uniquely and unambiguously characterize them, the short read length limits applicability for de novo sequencing. However, given the sequencing depth and the throughput of this instrument, stringent assembly of highly identical sequences can be achieved. We describe SSAKE, a tool for aggressively assembling millions of short nucleotide sequences by progressively searching through a prefix tree for the longest possible overlap between any two sequences. SSAKE is designed to help leverage the information from short sequence reads by stringently assembling them into contiguous sequences that can be used to characterize novel sequencing targets. Availability: http://www.bcgsc.ca/bioinfo/software/ssake.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes

alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.     

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current     

皮层神经发生的分子调控机制

在高等动物神经发育过程中,背侧端脑神经上皮细胞在多种调控基因及环境调控因子的作用下经历了错综复杂的发育事件,逐步形成具有精细的分层结构和高级功能的神经皮层。这些发育中的调控具有严格的时间和区域特性。本文主要就皮层形成中神经干细胞增殖、分化方向及皮层神经元亚型的特化等方面的调控机制予以论述。