Design, calibration, and performance of a uniaxial pressure cell for neutron scattering studies of quantum magnetism.

We provide an overview of a pressure cell designed to apply uniaxial pressure to single crystals for the study, by neutron scattering techniques, of strongly correlated magnetic systems and, in particular, quantum magnets. A detailed overview of the pressure cell components, their requirements, and links to the scientific and technical specifications are presented. The pressure cell is able to accommodate a 200 mm3 single crystal that can be pressurized up to 2 GPa at cryogenic temperatures. The pressure cell is consistent with the requirements of inelastic neutron scattering and, importantly, neutron polarization analysis. A particular strength of the uniaxial pressure cell is the highly uniform and low background for a wide scattering angle of 360° horizontally and ±20° vertically. We show the performance of the uniaxial pressure cell using a relevant neutron scattering instrument, the polarized diffuse scattering instrument, D7. The experiments confirm that the cell complies with the scientific and technical requirements. This uniaxial pressure cell will provide a useful additional tool in the sample environment suite available for the study of quantum magnetism.

Vitamin D and axonal injury in multiple sclerosis.

BACKGROUND: Previous studies in patients with multiple sclerosis (MS) have shown an association between high serum 25-hydroxyvitamin D (25[OH]D) levels and decreased inflammatory activity. OBJECTIVE: The purpose of this study was to examine the association between 25(OH)D levels and axonal injury in MS. Cerebrospinal fluid neurofilament light (CSF-NFL) was used as a marker for axonal injury. METHODS: Patients were identified through clinical practice at the Department of Neurology in Umeå University Hospital, Sweden. Blood draw, magnetic resonance imaging, scoring of disability and lumbar puncture were performed at inclusion in 153 patients, and also at median 12 months follow-up in 87 patients. For analyses of serum 25(OH)D levels and CSF-NFL, enzyme-linked immunosorbent assays were used. RESULTS: There was an inverse association between serum 25(OH)D and CSF-NFL levels in categorical (dichotomized at 75 or 100 nmol/l) analyses. A dose-response effect for 25(OH)D levels on CSF-NFL levels (p for trend=0.034) was also present. Serum 25(OH)D levels above 100 nmol/l were associated with lower CSF-NFL levels independently of ongoing MS treatment. CONCLUSION: High 25(OH)D levels are associated with decreased axonal injury in MS.

The roles of RgpB and Kgp in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83.

Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the gingipains in this VimA-dependent process, isogenic mutants defective in the gingipain genes in the vimA-deficient genetic background were evaluated. In contrast with the wild-type strain, RgpB and Kgp gingipain activities were absent in exponential phase in the ∆rgpA::tetQ-vimA::ermF mutant. However, these activities increased to 31 and 53%, respectively, of that of the wild-type during stationary phase. In the ∆rgpA::cat-∆kgp::tetQ-vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no gingipain activity observed in the ∆rgpB::cat-∆kgp::tetQ-vimA::ermF mutant whereas Kgp activity in ∆rgpA::cat-∆rgpB::tetQ-vimA::ermF mutant was 24% of the wild-type at late stationary phase. In contrast to RgpA, the glycosylation profile of the RgpB catalytic domain from both W83 and P. gingivalis FLL92 (vimA::ermF) showed similarity. Taken together, the results suggest multiple gingipain activation pathways in P. gingivalis. Whereas the maturation pathways for RgpA and RgpB are different, the late-onset gingipain activity in the vimA-defective mutant was due to activation/maturation of RgpB and Kgp. Moreover, unlike RgpA, which is VimA-dependent, the maturation/activation pathways for RgpB and Kgp are interdependent in the absence VimA.

(13)C cross-polarization/magic angle spinning NMR studies of alpha-elastin preparations show retention of overall structure and reduction of mobility with a decreased number of cross-links.

High-resolution solid-state (13)C NMR spectra are presented for samples of alpha-elastin prepared from the aorta of normal and copper-deficient pigs. Chemical shifts of the various peaks indicate that both the normal and undercross-linked peptides have similar overall structures. However, (13)C T(1), (13)C T(1 rho), and (1)H T(1 rho) measurements indicate that the alpha-elastin peptides obtained from the abnormal elastic fibers samples exhibit altered mobilities, particularly in their side chains. Results from spectra taken with a range of contact times and from dipolar dephasing experiments are consistent with conclusions reached with the relaxation measurements. Namely, the loss of function associated with the undercross-linked sample is correlated to a small but measurable difference in relative mobility.

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods.

Solid-state spectral editing techniques have been used by others to simplify 13C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance 13C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with alpha-elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical 13C CPMAS spectra. Spectral editing is particularly useful for simplifying he aliphatic region of the NMR spectrum of this elastin preparation.

Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.

Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation.

We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

A fast and simple method to calculate protonation states in proteins.

A simple model for electrostatic interactions in proteins, based on a distance and position dependent screening of the electrostatic potential, is presented. It is applied in conjunction with a Monte Carlo algorithm to calculate pK(alpha) values of ionizable groups in proteins. The purpose is to furnish a simple, fast, and sufficiently accurate model to be incorporated into molecular dynamic simulations. This will allow for dynamic protonation calculations and for coupling between changes in structure and protonation state during the simulation. The best method of calculating protonation states available today is based on solving the linearized Poisson-Boltzmann equation on a finite difference grid. However, this model consumes far too much computer time to be a practical alternative. Tests are reported for fixed structures on bacteriorhodopsin, lysozyme, myoglobin, and calbindin. The studies include comparisons with Poisson-Boltzmann calculations with dielectric constants 4 and 20 inside the protein, a model with uniform dielectric constant 80 and distance-dependent dielectric models. The accuracy is comparable to that of Poisson-Boltzmann calculations with dielectric constant 20, and it is considerably better than that with epsilon = 4. The time to calculate the protonation at one pH value is at least 100 times less than that of a Poisson-Boltzmann calculation. Proteins 1999;36:474-483.

General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation.

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.

Characterisation of STREAMLINE phenyl.

STREAMLINE Phenyl is a new hydrophobic interaction chromatography support designed for use in expanded bed adsorption. The phenyl groups are linked to STREAMLINE matrix via highly stable ether linkages. Within this development project the chemical and chromatographic stability as well as the breakthrough capacity for human IgG has been studied. The chemical stability was monitored as the carbon leakage from the matrix to the storage solution, pH 1-14 at 20 and 40 degrees C. The carbon content in the supernatant was determined with Total Organic Carbon (TOC) technique. In the chromatographic stability study STREAMLINE Phenyl was stored in eight different storage solutions under ambient conditions for 12 weeks and then tested in a chromatographic function test. The results show that the adsorbent is chemically stable and that the chromatographic properties are retained under the tested conditions. The breakthrough capacity study demonstrates the importance of the bed height for obtaining maximal dynamic capacity. Further, there is a good correlation between breakthrough data generated from packed bed and expanded bed runs.

Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

pK(a) calculations along a bacteriorhodopsin molecular dynamics trajectory.

Electrostatic calculations of pK(a-values) are reported along a 400 ps molecular dynamics trajectory of bacteriorhodopsin. The sensitivity of calculated pK(a) values to a number of structural factors and factors related to the modelling of the electrostatics are also studied. The results are very sensitive to the choice of internal dielectric constant of the protein (in the interval 2-4). Moreover it is important to include internal water molecules and to average over a long enough portion ( approximately 100 ps) of an equilibrium molecular dynamics trajectory. The internal waters are necessary to get an ion-counter ion complex with the Schiff base and Arg 82 protonated and the aspartic groups (85 and 212) deprotonated. The fluctuations along the MD-trajectory do not change the protonation state of internal residues at neutral pH. However, at other pH values the averaging along a trajectory maybe crucial to get correct protonation states. A relationship is found between the arginine group 82, the aspartic group 85 and the glutamate group 204. Glu 204 is protonated in the ground state but the pK(a) value decreases towards deprotonation when the chromophore isomerizes into the cis state.