A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.     

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

Arginine vasopressin produces inhibition upon respiration without pressor effect in the rat

The purpose of the current study was to examine where arginine vasopressin (AVP) inhibits respiration by direct action on the areas of the ventrolateral medulla (VLM) in the rat. The animal was anesthetized by urethane (1.2 g/kg, i.p.), paralyzed with gallamine triethiodide, and artificially ventilated. Catheterization of the femoral artery and vein, and bilateral vagotomy were performed. The rat was then placed upon a stereotaxic instrument in a prone position. The phrenic nerve was separated and cut peripherally. Phrenic nerve activity (PNA) was monitored at normocapnia and hypercapnia in hyperoxia. Microinjection of AVP into various subregions of the VLM was then performed. In response to AVP microinjection, a transient period of apnea and then a significant decrease in PNA amplitude were observed. Arterial blood pressure was unchanged. This inhibition of PNA with AVP treatment was site-specific, attenuated by raising CO2 concentration, and totally abolished by pretreatment with AVP V1A receptor antagonist. Data of the present study indicate that endogenous resource of AVP may produce an inhibitory effect upon respiration via AVP receptors presented on neurons within the VLM.     

Mechanistic aspects of CoII(HAPP)(TFA)2 in DNA bulge-specific recognition

A novel octahedral complex Co^II(HAPP)(TFA)₂ [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H₂O₂. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal–HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of Co^II(HAPP)(TFA)₂ was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3′-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for Co^IIHAPP²⁺ in the presence of H₂O₂ involves 4′-H abstraction of the deoxyribose moiety.     

An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays. fluorescence-activated cell sorting; green fluorescent protein; Ras small GTPases; anoikis     

The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose. Vid vesicles then deliver FBPase to the vacuole for degradation. A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole. For this assay, FBPase was fused with a truncated form of alkaline phosphatase. Under in vivo conditions, FBPase-delta60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation. Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-delta60Pho8p. These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system. Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner. The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles. In contrast, the t-SNARE Vam3p was a necessary vacuolar component. Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.     

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.     

Chromosomal polymorphisms due to heterochromatin growth and pericentric inversions in white-bellied rat,Niviventer confucianus,from China

Different cytogenetic techniques were used to analyze the chromosomes of white-bellied rat, Niviventer confucianus from Mt. Tai and Jinan, Shandong Province and Ningshan, Shaanxi Province of China. Shandong populations have 2n = 46 chromosomes with 4 metacentric, 2 subtelocentric, 16 telocentric pairs of autosomes and the submetacentric X and telocentric Y. The chromosomal arm number (NF) of the two populations was 56. Shaanxi population has 2n = 46 chromosomes with 4 metacentric, 1 submetacentric, 1 subtelocentric and 16 telocentric pairs of autosomes and the submetacentric X and telocentric Y. The karyotype of Ningshan population showed NF = 58. As the result of the comparison of C- and G-banding patterns, and compare with other species in the genus Niviventer, we suppose that the chromosomal evolution of Niviventer involved in pericentric inversion and heterochromatin growth. The submetacentric chromosomes of Shaanxi population would be originated from the growth of heterochromatin of the subtelocentric chromosome of Shandong population.