表达轮状病毒G2和G3型vp7基因重组腺病毒的免疫效果

为探索以非复制型腺病毒为表达载体的多价轮状病毒(Rotavirus,RV)基因工程疫苗的可行性,在前期工作的基础上,对表达我国G2和G3型RV流行毒株vp7基因的重组腺病毒的免疫效果进行了研究。分别用表达G2和G3型vp7基因的重组腺病毒rvAdG2VP7、rvAdG3VP7经滴鼻和灌胃两种途径免疫Balb/c小鼠,对免疫后小鼠的血清抗体、黏膜抗体和相关的细胞因子水平进行了检测和比较。结果表明,用表达G2和G3型vp7基因的重组腺病毒经滴鼻和灌胃两种途径免疫小鼠后,均可诱导机体产生较强的RV特异性免疫反应,包括体液免疫、细胞免疫和黏膜免疫,并能产生中和抗体。但免疫反应以Th2类为主,Th1类反应也占有相当的比例。本研究为新型RV基因工程疫苗的深入研究奠定了基础。     

Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage

AIMS: To determine the effectiveness of an alkaline cleaner used in food-processing plants and a lytic bacteriophage specific for Escherichia coli O157:H7 in killing wild type and rpoS-deficient cells of the pathogen in a biofilm. METHODS AND RESULTS: Wild type and rpoS-deficient cells were attached to stainless steel coupons (c. 7-8 log CFU per coupon) on which biofilms were developed during incubation at 22 degrees C for 96 h in M9 minimal salts media (MSM) with one transfer to fresh medium. Coupons were treated with 100 and 25% working concentrations of a commercial alkaline cleaner (pH 11.9, with 100 microg ml(-1) free chlorine) used in the food industry, chlorine solutions (50 and 100 microg ml(-1) free chlorine), or sterile deionized water (control) at 4 degrees C for 1 and 3 min. Treatment with 100% alkaline cleaners reduced populations by 5-6 log CFU per coupon, a significant (P < or = 0.05) reduction compared with treatment with water. Initial populations (2.6 log CFU per coupon) of attached cells of both strains were reduced by 1.2 log CFU per coupon when treated with bacteriophage KH1 (7.7 log PFU ml(-1)) for up to 4 days at 4 degrees C. Biofilms containing low populations (2.7-2.8 log CFU per coupon) of wild type and rpoS-deficient cells that had developed for 24 h at 22 degrees C were not decreased by more than 1 log CFU per coupon when treated with KH1 (7.5 log PFU ml(-1)) at 4 degrees C. CONCLUSIONS: Higher numbers of cells of E. coli O157:H7 in biofilms are killed by treatment with an alkaline cleaner than with hypochlorite alone, possibly through a synergistic mechanism of alkaline pH and hypochlorite. Populations of cells attached on coupons were reduced by treating with bacteriophage but cells enmeshed in biofilms were protected. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline pH, in combination with hypochlorite, in a commercial cleaner is responsible for killing E. coli O157:H7 in biofilms. Treatment with bacteriophage KH1 reduces populations of cells attached to coupon surfaces but not cells in biofilms.     

Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.     

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.