ELECTROPHORESIS OF STEROLS : III. FURTHER INVESTIGATIONS OF CHOLESTEROL SURFACES

1. No significant differences were noted between the electrophoretic mobilities of unground cholesterol crystallized from alcohol, acetone, or the melted state. 2. On grinding with ice at –10°C. the mobility drops to less than half (at pH 5.8) that observed in the unground state. This equilibrium condition is reached after 50 minutes grinding. Cholesterol crystals from alcohol or acetone behave identically throughout the course of this change. 3. When cholesterol crystals which have been ground with ice are dried in vacuo at room temperature they revert to the mobility of unground cholesterol. The cause of this phenomenon is discussed. 4. Both ground and unground cholesterol have an isoelectric point near pH 3.0, even after crystallization from the melted state.     

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     

Explaining Disparities in Use of Skilled Birth Attendants in Developing Countries: A Conceptual Framework

Despite World Health Organization recommendations that all women deliver with a skilled birth attendant (SBA), research continues to demonstrate large disparities in use of SBAs by socioeconomic status (SES). Yet few quantitative studies empirically examine the factors underlying these disparities, due in part to the fact that current models do not provide clear pathways—with measurable mediators—for how distal factors like SES may affect maternal health-seeking behaviors like delivering with SBAs. We propose the Disparities in Skilled Birth Attendance (DiSBA) framework for examining the determinants of use of SBAs. We posit that three proximal factors directly affect use of SBAs: perceived need, perceived accessibility of maternal health services, and perceived quality of care. Distal factors like SES affect use of SBAs indirectly through these proximal factors, and the effects can be measured. We test the assumptions of the DiSBA framework using data from the Ghana Maternal Health Survey. The analytic techniques we use include logistic regression with mediation analysis to examine the intervening effects. We find that our proxies for perceived access, perceived need, and perceived quality of care account for approximately 23% of the difference between women with no education and those with primary school education, and about 55% of the difference between women in the lowest wealth quintile and those in the middle wealth quintiles. This study suggests that proximal factors are worthy of increased attention in terms of measurement, data collection, analysis, programmatic efforts, and policy interventions, as these factors are potentially more amenable to change than the distal factors. The effects of proximal factors are also likely context specific, thus sufficient understanding in different contexts is essential to developing appropriate interventions.     

Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro.

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.     

Structural changes in the murine yolk sac during gestation: cytochemical and electron microscope observations

Specimens of murine yolk sac prepared for light and electron microscopy at 8, 12, and 18 days of gestational age revealed a changing morphology of the endodermal cells in the visceral layer as gestation progressed. Basophilia increased between 8 and 12 days and decreased slightly between 12 and 18 days. These changes corresponded to the development of a highly organized rough-surfaced endoplasmic reticulum by 12 days and its partial replacement by smooth-surfaced elements by 18 days. Staining of frozen sections with tetrazolium dyes revealed a substrate dependent localization of the staining reaction within the cells and suggested an underlying compartmentalization of metabolic activities. Two types of apical granules were observed on the basis of both cytochemistry and electron microscopy. These granules were most numerous at 12 days of gestational age. The observations suggest that although synthesis and absorption doubtless occur simultaneously throughout gestation, the emphasis is on synthesis during the first half of the period and absorption during the second half. It is suggested that the change in fine structure may be due to the rupture of Reichert's membrane and the subsequent establishment of direct contact between the endodermal cells and the uterine wall.     

Testing the Hypothesis of Biofilm as a Source for Soft Tissue and Cell-Like Structures Preserved in Dinosaur Bone

Recovery of still-soft tissue structures, including blood vessels and osteocytes, from dinosaur bone after demineralization was reported in 2005 and in subsequent publications. Despite multiple lines of evidence supporting an endogenous source, it was proposed that these structures arose from contamination from biofilm-forming organisms. To test the hypothesis that soft tissue structures result from microbial invasion of the fossil bone, we used two different biofilm-forming microorganisms to inoculate modern bone fragments from which organic components had been removed. We show fundamental morphological, chemical and textural differences between the resultant biofilm structures and those derived from dinosaur bone. The data do not support the hypothesis that biofilm-forming microorganisms are the source of these structures.     

Cloning and functional expression of the cytoplasmic form of rat aminopeptidase P

A rat cytoplasmic aminopeptidase P was purified from liver cytosol with a procedure including an affinity elution step with 3 microM inositol 1,3,4-trisphosphate. Proteolytic fragments were generated, sequenced and the enzyme was cloned from a rat liver cDNA library. The structure shows high (87.8% and 95.5%, respectively) sequence identity at the nucleotide and amino acid levels with the previously described human putative cytoplasmic aminopeptidase P. The cloned rat enzyme was functionally expressed in Escherichia coli and also in COS-1 cells. Western blot analysis, using an antibody generated against the recombinant protein, and Northern blot hybridization showed ubiquitous expression of the protein in different tissues with the highest expression level in the testis.     

Neuromuscular and psychological influences on range of motion recovery in anterior cruciate ligament reconstruction patients

To identify distinguishing characteristics for knee surgery patients who experience a protracted recovery process, we sought to determine if there is an association between the neuromuscular stretch reflex and psychological factors of pain perception and anxiety on the range of motion (ROM) recovery rate of post-operative anterior cruciate ligament reconstruction (ACLR) rehabilitation patients. The ACLR participants were categorized into a slow recovery group (SRG: >6 weeks to recover 0–125° knee flexion [n = 10]) and a normal recovery group (NRG: <6 weeks to recovery 0–125° knee flexion [n = 12]). Control participants (n = 22) were age, gender and activity-level matched to the surgical participants. Neuromuscular testing consisted of sagittal plane video kinematics of the Wartenberg Pendulum Test for determining lower limb stiffness indices and electromyography-monitored patellar tendon tap reflex responses. Psychological and health status assessments consisted of the State–Trait Anxiety Inventory and SF-36? Health Survey. Data revealed that neuromuscular reflex profiles, lower limb stiffness indices, pain, anxiety and SF-36? indices of function were not significantly different between the two surgical groups (SRG and NRG). The surgical groups exhibited significantly greater pain (2.67 ± 2.27 SRG, 1.49 ± 1.15 NRG) than the control group (p ? .05). SF-36? indices were significantly lower for the surgical groups for total score (546.55 ± 94.70 SRG, 577.57 ± 125.58 NRG), function 69.00 ± 20.24 SRG, 67.08 ± 19.12 NRG), role physical (53.75 ± 22.85 SRG, 53.12 ± 23.15 NRG), social (76.24 ± 25.31 SRG, 65.62 ± 27.24 NRG), and emotional (82.49 ± 19.81 SRG, 81.38 ± 23.02 NRG) subscales (p ? .05). These results suggest that neuromuscular reflex responses, visual analogue scale (VAS) pain, and anxiety are not distinguishing factors for ROM recovery rate between the SRG and NRG. Decreased SF-36? indices, including pain as it influences function, though clinically relevant factors, were not statistically associated with post-operative ROM recovery rate.