Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of 相似文献   

Peptide nucleic acids and their phosphonate analogues: synthesis and hybridization characteristics

The synthesis of a series of DNA mimics--peptide nucleic acids, phosphonate analogues of peptide nucleic acids, and their hybrids--is described. The preparative synthesis of the corresponding monomers and the solid phase automated synthesis of oligomers-mimics are developed. Modified phosphonate analogues of peptide nucleic acids, in particular chiral derivatives and those with additional hydroxyl groups in the side chains of the backbone as well as pyrene derivatives of peptide nucleic acids and their phosphonate analogues, are prepared. The ability of the resulting oligomers specifically to hybridize to DNA and RNA complementary chains is studied. It is shown that phosphonate analogues of peptide nucleic acids and their hybrids with peptide nucleic acids can form complexes with the DNA and RNA complementary strands, the stability of the complexes increasing in parallel with the increase in the number of peptide nucleic acid residues in the chain of the mimic. This property, along with good water solubility, provides the precondition for further evaluation of these compounds as antisense and antigene agents.     

The fate of pelvic osteolysis after reoperation. No recurrence with lesional treatment

Twenty-three hips (21 patients) with 30 pelvic osteolytic lesions underwent reoperation and were observed prospectively for 25 to 74 months (average, 40 months) to assess the fate of pelvic osteolysis after reoperation. The average radiographic dimensions of the lytic lesions were 2.4 x 1.9 cm with the largest lesion measuring 7 x 5 cm. The porous ingrowth acetabular component shell had been left in situ in 15 hips and had been revised in eight. There was no difference in the average lesional size for hips with revised shells compared with those with unrevised shells. In cases where the shell was left in situ, the osteolytic lesions were curetted by working around the component perimeter or through holes in the shell. In 18 hips the bone defect(s) were grafted with autograft or allograft. Regardless of the management of the acetabular shell or the absence or presence of bone graft, none of the osteolytic lesions have progressed. Twenty-six of the 30 lesions have increased radiographic density. All acetabular components remain radiographically well fixed. There were no new osteolytic lesions. All hips were functioning well, and none have required subsequent reoperation for any reason. There was a statistically significant reduction in the operative time and the amount of blood loss when the acetabular component was not revised. It does not appear necessary to remove a well fixed and well positioned cementless acetabular component for the treatment of pelvic osteolysis. Debridement of periarticular inflammatory tissue and lesional curettage, either with or without bone graft, is effective in managing this type of bone loss. Revision of the acetabular component shell was associated with a significant increase in operative time and blood loss. These results support routine radiographic evaluation after total hip arthroplasty to monitor the development of osteolysis. On the basis of this experience, the authors recommend lesional treatment of progressive pelvic osteolysis to avoid more difficult surgery and minimize patient morbidity.     

Systematic, computer-assisted optimisation of the isolation of Thermus thermophilus 50S ribosomal proteins by reversed-phase high-performance liquid chromatography

Isolation of the 50S ribosomal proteins from Thermus thermophilus has been achieved for the first time using reversed-phase high-performance liquid chromatography based on the use of the non-end-capped LiChrospher RP-18 sorbent and computer-assisted method development for optimisation of the resolution. The separation approach for these basic ribosomal proteins utilised mobile phases of high ionic strength, to suppress silanophilic interactions with this type of reversed-phase sorbent. These conditions were found to be a key requirement for achieving good resolution with minimal peak-tailing. The retention times of the 50S ribosomal proteins of Thermus thermophilus were observed to be in very close agreement with the values predicted by computer simulation procedures based on linear solvent strength concepts, with an average error of only 0.5%, whilst base-line resolution was achieved for most of the adjacent peak zones. Following N-terminal sequencing, the proteins TthL5, TthL9, TthL18, TthL24, TthL29, TthL32, TthL34, TthL35 and TthL36 of Thermus thermophilus were readily identified. This approach thus provided a readily optimised strategy for the isolation of the 50S ribosomal proteins from Thermus thermophilus and should be generally applicable to the corresponding ribosomal proteins from various other species, as well as other classes of basic proteins present in crude extracts derived from other biological sources.     

Effects of curvature and stenosis-like narrowing on wall shear stress in a coronary artery model with phasic flow

To gain insight into the details of intracoronary flow we have used computational fluid dynamic techniques to determine the velocity and wall shear stress distributions in both steady- and phasic-flow models of a curved coronary artery with several degrees of stenosis. The steady-flow Reynolds number was 500 and the peak phasic flow Reynolds number was 700. Without stenosis and at 25% (area) stenosis wall shear stress and velocities are higher at the outer wall than the inner wall but retain the same direction as the superimposed flow. At higher stenoses laminar flow separation occurs and the inner wall is exposed to shear stresses that vary widely, both temporally and spatially.     

Differential diagnosis of posterior cranial fossa: potentialities and unsolved problems

This study describes nurse practitioners' (NPs) practice and employment trends, primary care/direct patient care involvement, scope of practice issues, productivity, and demographic characteristics. Data were collected by mailed survey from 2,499 NPs in New York State. NPs were predominantly certified as Adult, Pediatric or Family NPs. The vast majority of NPs were working (64.1% full-time; 22.7% part-time). Fifty percent said they spend over 80% of their time providing primary care. Over 50% of the NPs employed in non-hospital settings said that their organizations had contracts with four or more managed care organizations.     

Obliteration of gallbladder lumen in patients with high operation risk

The paper presents pilot results of clinical application of developed experimintally method of treatment for acute cholecystitis based on demucosation of the gall-bladder followed by obliteration of its lumen. Two modifications of the method-treatment of mucous membrane of the gall-bladder by phenol emulsion (16 patients) and electrocoagulation of the mucosa (49 patients)--were used with good clinical results in patients with high operation risk. Follow-up results are available for up to 3.5 years.     

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene

1,3-Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixture of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene.