Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

The effect of heavy- vs. light-load jump squats on the development of strength, power, and speed

The purpose of this investigation was to examine the effect of an 8-week training program with heavy- vs. light-load jump squats on various physical performance measures and electromyography (EMG). Twenty-six athletic men with varying levels of resistance training experience performed sessions of jump squats with either 30% (JS30, n = 9) or 80% (JS80, n = 10) of their one repetition maximum in the squat (1RM) or served as a control (C, n = 7). An agility test, 20-m sprint, and jump squats with 30% (30J), 55% (55J), and 80% (80J) of their 1RM were performed before and after training. Peak force, peak velocity (PV), peak power (PP), jump height, and average EMG (concentric phase) were calculated for the jumps. There were significant increases in PP and PV in the 30J, 55J, and 80J for the JS30 group (p 相似文献   

Innate differences in protein expression in the nucleus accumbens and hippocampus of inbred alcohol-preferring and -nonpreferring rats

Two-dimensional gel electrophoresis (2-DE) was used to separate protein samples solubilized from the nucleus accumbens and hippocampus of alcohol-na?ve, adult, male inbred alcohol-preferring (iP) and alcohol-nonpreferring (iNP) rats. Several protein spots were excised from the gel, destained, digested with trypsin, and analyzed by mass spectrometry. In the hippocampus, 1629 protein spots were matched to the reference pattern, and in the nucleus accumbens, 1390 protein spots were matched. Approximately 70 proteins were identified in both regions. In the hippocampus, only 8 of the 1629 matched protein spots differed in abundance between the iP and iNP rats. In the nucleus accumbens, 32 of the 1390 matched protein spots differed in abundance between the iP and iNP rats. In the hippocampus, the abundances of all 8 proteins were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins were higher in the iNP than iP rat. In the hippocampus, only 2 of the 8 proteins that differed could be identified, whereas in the nucleus accumbens 21 of the 32 proteins that differed were identified. Higher abundances of cellular retinoic acid-binding protein 1 and a calmodulin-dependent protein kinase (both of which are involved in cellular signaling pathways) were found in both regions of the iNP than iP rat. In the nucleus accumbens, additional differences in the abundances of proteins involved in (i) metabolism (e.g., calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (ii) cyto-skeletal and intracellular protein transport (e.g., beta-actin), (iii) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (iv) cellular signaling pathways (e.g., protein kinase C-binding protein), (v) synaptic function (e.g., complexin I, gamma-enolase, syndapin IIbb), (vi) reduction of oxidative stress (thioredoxin peroxidase), and (vii) growth and differentiation (hippocampal cholinergic neurostimulating peptide) were found. The results of this study indicate that selective breeding for disparate alcohol drinking behaviors produced innate alterations in the expression of several proteins that could influence neuronal function within the nucleus accumbens and hippocampus.     

Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division

Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to Escherichia coli ftsX. Two of the remaining 16 filamentous mutants also carried insertions in ftsX. Introduction of the wild-type F. johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In E. coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in F. johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.     

The structure and oligomerization of the yeast arginine methyltransferase, Hmt1

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.     

The extent of linkage disequilibrium in four populations with distinct demographic histories

The design and feasibility of whole-genome-association studies are critically dependent on the extent of linkage disequilibrium (LD) between markers. Although there has been extensive theoretical discussion of this, few empirical data exist. The authors have determined the extent of LD among 38 biallelic markers with minor allele frequencies >.1, since these are most comparable to the common disease-susceptibility polymorphisms that association studies aim to detect. The markers come from three chromosomal regions-1,335 kb on chromosome 13q12-13, 380 kb on chromosome 19q13.2, and 120 kb on chromosome 22q13.3-which have been extensively mapped. These markers were examined in approximately 1,600 individuals from four populations, all of European origin but with different demographic histories; Afrikaners, Ashkenazim, Finns, and East Anglian British. There are few differences, either in allele frequencies or in LD, among the populations studied. A similar inverse relationship was found between LD and distance in each genomic region and in each population. Mean D' is.68 for marker pairs <5 kb apart and is.24 for pairs separated by 10-20 kb, and the level of LD is not different from that seen in unlinked marker pairs separated by >500 kb. However, only 50% of marker pairs at distances <5 kb display sufficient LD (delta>.3) to be useful in association studies. Results of the present study, if representative of the whole genome, suggest that a whole-genome scan searching for common disease-susceptibility alleles would require markers spaced < or = 5 kb apart.     

Feedback control of mesolimbic somatodendritic dopamine release in rat brain

The objective of this study was to examine the role of dopamine (DA) receptors in the nucleus accumbens (ACB) in controlling feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats using ipsilateral dual-probe in vivo microdialysis. Perfusion of the ACB for 60 min with the DA uptake inhibitor GBR-12909 (10-1,000 microM) or nomifensine (10-1,000 microM) dose-dependently increased the extracellular levels of DA in ACB and concomitantly reduced the extracellular levels of DA in the VTA. Coperfusion of 100 microM nomifensine with either 100 microM SCH-23390 (SCH), a D1 antagonist, or 100 microM sulpiride (SUL), a D2 receptor antagonist, produced either an additive (for SCH) or a synergistic (for SUL) elevation in the extracellular levels of DA in the ACB, whereas the reduction in the extracellular levels of DA in the VTA produced by nomifensine alone was completely prevented by addition of either antagonist. Application of 100 microM SCH or SUL alone through the microdialysis probe in the ACB increased the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA remained unchanged. Overall, the results suggest that (a) increasing the synaptic levels of DA in the ACB activates a long-loop negative feedback pathway to the VTA involving both D1 and D2 postsynaptic receptors and (b) terminal DA release within the ACB is regulated directly by D2 autoreceptors and may be indirectly regulated by D1 receptors, possibly on interneurons and/or through postsynaptic inhibition of the negative feedback loop.     

Small synthetic ligands of the cholecystokinin-B/gastrin receptor can mimic the function of endogenous peptide hormones.

The gastric cholecystokinin-B/gastrin receptor (CCK-BR) is a key regulator of enterochromaffin-like cell function and proliferation. Over the last decade, a number of small non-peptide CCK-BR "antagonists" have been discovered. Here, we demonstrate that some of these non-peptide ligands in fact possess significant ability to activate the human CCK-BR, and are, therefore, more properly categorized as partial agonists. When tested in COS-7 cells transiently expressing the recombinant human CCK-BR, saturating concentrations of the small "peptoid" ligands PD 135,158 and PD 136,450 stimulated inositol phosphate formation to 23 and 43 percent, respectively, of the maximum response induced by a considerably larger endogenous peptide agonist, cholecystokinin octapeptide. In contrast, the benzodiazepine-derived CCK-BR ligand, YM022, acted as a "true" high-affinity antagonist of cholecystokinin-induced inositol phosphate formation (pA2 = 9.69). Consistent with recent findings in animal experiments, our data reveal that small synthetic ligands have the potential to function as either CCK-BR agonists or antagonists. These dual properties of synthetic molecules must be considered when evaluating candidate drugs for human disease.