All-Order Full-Coulomb Quantum Spectral Line-Shape Calculations.

Understanding how atoms interact with hot dense matter is essential for astrophysical and laboratory plasmas. Interactions in high-density plasmas broaden spectral lines, providing a rare window into interactions that govern, for example, radiation transport in stars. However, up to now, spectral line-shape theories employed at least one of three common approximations: second-order Taylor treatment of broadening operator, dipole-only interactions between atom and plasma, and classical treatment of perturbing electrons. In this Letter, we remove all three approximations simultaneously for the first time and test the importance for two applications: neutral hydrogen and highly ionized magnesium and oxygen. We found 15%-50% change in the spectral line widths, which are sufficient to impact applications including white-dwarf mass determination, stellar-opacity research, and laboratory plasma diagnostics.

Correlating results from high resolution EBSD with TEM- and ECCI-based dislocation microscopy: Approaching single dislocation sensitivity via noise reduction.

High resolution electron backscatter diffraction (HREBSD), an SEM-based diffraction technique, may be used to measure the lattice distortion of a crystalline material and to infer the geometrically necessary dislocation content. Uncertainty in the image correlation process used to compare diffraction patterns leads to an uneven distribution of measurement noise in terms of the lattice distortion, which results in erroneous identification of dislocation type and density. This work presents a method of reducing noise in HREBSD dislocation measurements by removing the effect of the most problematic components of the measured distortion. The method is then validated by comparing with TEM analysis of dislocation pile-ups near a twin boundary in austenitic stainless steel and with ECCI analysis near a nano-indentation on a tantalum oligocrystal. The HREBSD dislocation microscopy technique is able to resolve individual dislocations visible in TEM and ECCI and correctly identify their Burgers vectors.

Host associations of Dermacentor, Amblyomma, and Ixodes (Acari: Ixodidae) ticks in Tennessee.

From April 2007 to September 2008, 1,793 adult and nymphal ixodid ticks were collected from 49 counties in Tennessee. Six species were identified, including Dermacentor variabilis (Say), Amblyomma americanum (L.), Ixodes texanus (Banks), Ixodes cookei Packard, Ixodes scapularis (Say), and Amblyomma maculatum Koch, from 13 medium- to large-sized mammalian hosts and dragging through vegetation. Raccoons were the most common vertebrate source (198 captures), accounting for 60% of ticks collected. Dermacentor variabilis was the predominant species from raccoons with a prevalence of 92% and mean intensity of 5.3. A. americanum was predominated in white-tailed deer and drags with respective mean intensities of 3.1 and 14.1 and prevalence values of 94%. All tick species were identified between April and August, coinciding with the majority of animal captures. Only A. americanum, I. texanus, and I. cookei were identified from 22 animal captures from November to March. I. texanus and I. cookei were more common in the eastern portions of the state, but this may be a result of higher raccoon captures in those areas. Only four specimens of I. scapularis were collected in this study, which may reflect the absence of small mammal or reptile captures. Two A. maculatum were collected, and we report new distribution records in Tennessee for this species. Despite unequal sampling among ecoregions, the large numbers of D. variabilis and A. americanum from multiple host species suggest their widespread distribution throughout the state. These species of ticks can transmit multiple pathogens, including spotted fever group rickettsiae and ehrlichiae.

Load-carrying capacity of the human cervical spine in compression is increased under a follower load.

STUDY DESIGN: An experimental approach was used to test human cadaveric cervical spine specimens. OBJECTIVE: To assess the response of the cervical spine to a compressive follower load applied along a path that approximates the tangent to the curve of the cervical spine. SUMMARY OF BACKGROUND DATA: The compressive load on the human cervical spine is estimated to range from 120 to 1200 N during activities of daily living. Ex vivo experiments show it buckles at approximately 10 N. Differences between the estimated in vivo loads and the ex vivo load-carrying capacity have not been satisfactorily explained. METHODS: A new experimental technique was developed for applying a compressive follower load of physiologic magnitudes up to 250 N. The experimental technique applied loads that minimized the internal shear forces and bending moments, loading the specimen in nearly pure compression. RESULTS: A compressive vertical load applied in the neutral and forward-flexed postures caused large changes in cervical lordosis at small load magnitudes. The specimen collapsed in extension or flexion at a load of less than 40 N. In sharp contrast, the cervical spine supported a load of up to 250 N without damage or instability in both the sagittal and frontal planes when the load path was tangential to the spinal curve. The cervical spine was significantly less flexible under a compressive follower load compared with the hypermobility demonstrated under a compressive vertical load (P < 0.05). CONCLUSION: The load-carrying capacity of the ligamentous cervical spine sharply increased under a compressive follower load. This experiment explains how a whole cervical spine can be lordotic and yet withstand the large compressive loads estimated in vivo without damage or instability.

Mycophenolic acid increases apoptosis, lysosomes and lipid droplets in human lymphoid and monocytic cell lines.

BACKGROUND: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, is the active agent of the immunosuppressive drug, mycophenolate mofetil (MMF). Previous studies have shown that MPA inhibits DNA synthesis in T and B lymphocytes by blocking de novo guanosine synthesis, and that MPA induces monocyte differentiation. MMF is being used for prevention of organ graft rejection and has also shown efficacy in rheumatoid arthritis trials. This study was designed to determine if apoptosis also plays a role in the immunosuppressive and anti-inflammatory effects of MMF. METHODS: Cultured human T lymphocytic (MOLT-4) and monocytic (THP-1 and U937) cell lines were treated with MPA. Apoptosis, cell viability, DNA content, lipid content, cell volume, and lysosomes were measured by a variety of microscopic, flow cytometric, and biochemical techniques. RESULTS: MPA inhibits proliferation, arrests cell cycle in S phase, and increases apoptosis in all three cell lines. Exogenous guanosine added within 24 hr of MPA treatment, but not later, partially reversed MPA-induced apoptosis in MOLT-4 cells. MPA increased lipid droplets in all three cell lines and increased both cell volumes and numbers of lysosomes in the monocytic cell lines. In both monocytic cell lines, MPA also reduced the number of nuclei containing nucleoli and greatly increased neutral lipids, primarily triacylglycerols, suggesting that these cells were differentiating. CONCLUSIONS: Increased apoptosis and terminal differentiation of both lymphocytes and monocytes may promote the antiproliferative, immunosuppressive, and anti-inflammatory effects of MMF seen clinically in transplantation and rheumatoid arthritis.

A follower load increases the load-carrying capacity of the lumbar spine in compression.

STUDY DESIGN: An experimental approach was used to test human cadaveric spine specimens. OBJECTIVE: To assess the response of the whole lumbar spine to a compressive follower load whose path approximates the tangent to the curve of the lumbar spine. SUMMARY OF BACKGROUND DATA: Compression on the lumbar spine is 1000 N for standing and walking and is higher during lifting. Ex vivo experiments show it buckles at 80-100 N. Differences between maximum ex vivo and in vivo loads have not been satisfactorily explained. METHODS: A new experimental technique was developed for applying a compressive follower load of physiologic magnitudes up to 1200 N. The experimental technique applied loads that minimized the internal shear forces and bending moments, made the resultant internal force compressive, and caused the load path to approximate the tangent to the curve of the lumbar spine. RESULTS: A compressive vertical load applied in the neutral lordotic and forward-flexed postures caused large changes in lumbar lordosis at small load magnitudes. The specimen approached its extension or flexion limits at a vertical load of 100 N. In sharp contrast, the lumbar spine supported a load of up to 1200 N without damage or instability when the load path was tangent to the spinal curve. CONCLUSIONS: Until this study, an experimental technique for applying compressive loads of in vivo magnitudes to the whole lumbar spine was unavailable. The load-carrying capacity of the lumbar spine sharply increased under a compressive follower load, as long as the load path remained within a small range around the centers of rotation of the lumbar segments. The follower load path provides an explanation of how the whole lumbar spine can be lordotic and yet resist large compressive loads. This study may have implications for determining the role of trunk muscles in stabilizing the lumbar spine.

Self-reactive B lymphocytes overexpressing Bcl-xL escape negative selection and are tolerized by clonal anergy and receptor editing.

Self-reactive B cells Tg for both a bcl-xL death inhibitory gene and an Ig receptor recognizing hen egg lysozyme (HEL-Ig) efficiently escaped developmental arrest and deletion in mice expressing membrane-bound self-antigen (mHEL). In response to the same antigen, Tg HEL-Ig B cells not expressing bcl-xL were deleted, while cells expressing bcl-2 were arrested at the immature B stage. Bcl-xL Tg B cells escaping negative selection were anergic in both in vitro and in vivo assays and showed some evidence for receptor editing. These studies suggest that Bcl-x may have a distinct role in controlling survival at the immature stage of B cell development and demonstrate that tolerance is preserved when self-reactive B cells escape central deletion.

The need for individualized dosimetry for tangential breast treatment.

We have examined the isodose distributions of 119 intact breast patients treated on a 6 MV linac to determine if a library of treatment plans could be used instead of individualized computer plans for patient treatments without compromising the quality of those treatments. The parameters studied were: field width, baseline separation, central axis separation, wedge angle, and isodose coverage. At least two wedges were used in the computer plans for each patient and the best plan was then chosen. In order to construct a library of plans, the choice of wedge, treatment isodose, and dose uniformity should be predictable. Our results show that for 90 out of 119 plans (76%), the 30 degrees wedge was best. In the other 29 cases, either the 15 degrees or the 45 degrees wedge yielded better plans. On average, the improvement in dose homogeneity due to choice of wedge was about 2% (range 0-7%) for these cases. Although grouping like-patient parameters generally restricted the isodose variation to +/- 2.5%, there were five patients for which up to a 7% underdosage would not have been predicted. For the set of plans using a 30 degrees wedge, a significant correlation was found for the ratio of the baseline to central axis separation vs. treatment isodose. The average isodose which covered the target area was 97% (range 90-100%) and 102 out of 107 patient plans using the 30 degrees wedge fell between 94 and 100%. We conclude from these results that the variation in dose distribution found with seemingly similar sized breasts is due to the variation in breast shape and symmetry. The use of a library plan with a single wedge and a standardized isodose line for tangential field treatment of intact breast could cause up to a 7% dose difference compared to the actual dosimetry for that patient.

Azalanstat (RS-21607), a lanosterol 14 alpha-demethylase inhibitor with cholesterol-lowering activity.

Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14 alpha-demethylase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dose-dependent manner (ED50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering (r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50-75 mg/kg) stimulated hepatic microsomal cholesterol 7 alpha-hydroxylase activity by 50-400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis.

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.

Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.

Synthesis and immunosuppressive activities of conformationally restricted cyclosporin lactam analogues.

Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, is known to adopt predominantly a single conformation in chloroform solution, characterized in part by a type II' beta-turn encompassing Abu-Sar-MeLeu-Val (residues 2-5). In order to evaluate whether this beta-turn is bound by the receptor, we previously had prepared a conformationally restricted beta-turn analogue, (delta-lactam) CsA (2), which was found to retain only weak immunosuppressive activity, a result that could indicate that steric hindrance between receptor and the lactam atoms in 2 diminished activity or that the type II' beta-turn is not a feature in the bioactive conformation of CsA. In an attempt to distinguish between these two possibilities, we have synthesized two new CsA analogues, (gamma-lactam) CsA (3) and (des-N-methyl-lactam) CsA (4), which contain less sterically demanding conformational restrictions. The immunosupressive activity of each analogue (4-13% and 7-17%, respectively, relative to CsA), measured in an assay that determined the inhibition of concanavalin A stimulated thymocytes, is essentially equipotent with that of the delta-lactam. The chemical shifts and temperature dependencies of the protons in analogues 3 and 4 are very similar to the corresponding protons in CsA and in 2, which suggest that the solution conformations of the small lactam analogues are very similar to that of the delta-lactam 2. The synthesis of the lactam components and the corresponding CsA derivatives is described. Reduction in the size of the lactam ring does not lead to enhanced immunosuppressive activity.

Synthesis, conformation, and immunosuppressive activity of cyclosporines that contain epsilon-oxygen (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine analogues in the 1-position.

A series of CsA analogues that contain novel epsilon-oxygen isosteres of (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine (MeBmt) in the 1-position were synthesized. The key steps for the syntheses of enantiomerically pure epsilon-oxygen MeBmt analogues 4-7 were based on the stereoselective epoxidation of cis-allylic alcohol derivative 12 with a peracid, followed by the application of a base-catalyzed intramolecular rearrangement of epoxyurethane 15, which was derived from the reaction of epoxy alcohol 14 and methyl isocyanate. All epsilon-oxygen MeBmt analogues have the same stereochemistry and the same functional groups as those on the alpha,beta,gamma-carbons of MeBmt except for the double bond of MeBmt, which is replaced by the -OCH2-group. The syntheses of the peptide portion of CsA analogues followed the strategy we reported previously. The immunosuppressive activities of CsA analogues 28a-e, determined by inhibition of concanavalin A stimulated thymocytes, showed that 28b, which has the closest structural resemblance to MeBmt, retains about 7-10% of activity of CsA, whereas the analogues 28a, 28c, and 28e retain about 2-5% activity. It is interesting to note that 28d, which has the larger benzyl group on the end of the side chain, is about 20-25% as active as CsA. Extensive conformational analyses by 1D and 2D NMR indicated that the conformation of the 33-membered peptide ring system for all CsA analogues was very similar to that of CsA. However, the NMR analyses revealed that the 1-position side chain of all these CsA analogues adopted a novel conformation in chloroform by forming a different intramolecular hydrogen bond between the beta-OH and the epsilon-oxygen of the same residue. The NMR data also suggest that the chloroform conformation of these CsA analogues is similar to the conformation observed in the crystal structure of CsA in that the 1-position side chain is folded across the cyclic peptide ring system.

Synthesis, conformation, and immunosuppressive activities of three analogues of cyclosporin A modified in the 1-position.

The syntheses of three new cyclosporin A (CsA) analogues that contain novel MeBmt derivatives in the 1-position are described. The MeBmt analogue that contains an additional methyl group on C4, (2S,3R,6E)-4,4-dimethyl-3-hydroxy-2-(N-methylamino)-6-octenoic acid (MeBm2t), was synthesized in four steps beginning with the reaction of Pmz-Sar-OtBu with (4E)-2,2-dimethyl-4-hexenal. The C4 desmethyl analogue of MeBmt, (2S,3R,6E)-3-hydroxy-2-(N-methylamino)-6-octenoic acid (MeBth), was synthesized in nine steps by a route based on the Sharpless chiral epoxidation procedure. The alkynyl derivative of MeBmt, (2S,3R,4R)-4-methyl-3-hydroxy-2-(N-methylamino)-6-octynoic acid (MeByt), was synthesized by a modification of the procedure described by Tung et al. for the synthesis of MeBmt. Each MeBmt analogue was protected as the N,O-acetonide and coupled with the hexapeptide Abu-Sar-MeLeu-Val-MeLeu-Ala-OBzl. The resulting heptapeptide was deprotected and coupled with Fmoc-D-Ala-MeLeu-MeLeu-MeVal-OH. The resulting undecapeptides were deprotected and cyclized to give the corresponding CsA analogues. Conformational analysis by 1D and 2D NMR methods was carried out for each analogue in chloroform, and the results are compared with the corresponding solution conformations of CsA and dihydrocyclosporin. The immunosuppressive activities of each analogue, determined in concanavalin A stimulated thymocytes, are lower than obtained for CsA. The results establish the important effect the methyl group and the double bond in MeBmt have on the solution conformation of the 1-position residue in CsA and on immunosuppressive activity.

Synthesis, biological activity, and conformational analysis of (2S,3R,4S)-MeBmt1-cyclosporin, a novel 1-position epimer of cyclosporin A.

Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, contains a unique amino acid, (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine (MeBmt), that appears to be critically involved in the biological activity of CsA. In order to further explore the effect that structural elements in MeBmt have on the conformation and biological activity of CsA, the 4-epimer of MeBmt [(4S)-MeBmt, 2] and the corresponding CsA analogue [(4S)-MeBmt1-CsA, 3] have been synthesized. Biological assay using concanavalin A stimulated thymocytes indicated that (4S)-MeBmt1-CsA (3) has only 2-4% immunosuppressive activity relative to CsA. The NMR analysis by 1D and 2D NMR methods establishes the conformation of 3, of which the 33-membered cyclic peptide ring system in chloroform is very similar to that of CsA. However, the NMR analysis also reveals that the 1-position side chain orientation in (4S)-MeBmt1-CsA (3) is very different from that of CsA. Specifically, the (4S)-MeBmt alpha,beta-torsion angle (chi 1) has been rotated approximately 120 degrees relative to that of CsA, and the orientation of the butenyl side chain relative to the 33-membered peptide backbond is different. The orientation of the (4S)-MeBmt side chain is consistent with the possible conformations calculated for (4S)-MeBmt1-CsA (3) by using molecular mechanics (in vacuo) calculations. The conformational analysis suggests that the loss of biological activity for 3 results from an altered conformation of the 1-position side chain relative to the peptide backbond due to the changed chirality at C4 of MeBmt.

Synthesis, conformation, and immunosuppressive activity of a conformationally restricted cyclosporine lactam analogue.

Cyclosporine A (CsA, 1), an immunosuppressive cyclic undecapeptide, in apolar solvents adopts a II' beta-turn at the Sar3-MeLeu4 residues. [D-Proline3]Cs has been reported to be a nonimmunosuppressive analogue in which the II' beta-turn is retained. In order to determine if this loss of activity is caused by steric hindrance between the Cs analogue and its receptor or is caused by a change in the peptide conformation, an analogue that stabilizes a II' beta-turn has been synthesized, [lactam3,4]Cs. We also have studied the solution conformation of two other analogues, [D-MeAla3]Cs and [L-MeAla3]Cs. The conformations have been established by 1D difference NOE and 2D (NOESY or ROESY) NMR. The conformations of [lactam3,4]Cs and [D-MeAla3]Cs are indistinguishable from that of CsA in solution. [L-MeAla3]Cs was found to adopt a conformation with a cis amide bond between Sar3 and MeLeu4. The inhibition of concanavalin A stimulated thymocytes by CsA, [D-MeAla3]Cs, [L-MeAla3]Cs, and [lactam3,4]Cs gave IC50 values (nM) of 5, 6, 100, and 100, respectively. The weak immunosuppressive activity of [lactam3,4]Cs possessing the II' beta-turn suggests that the loss of activity for 4 is due to steric hindrance with the Cs receptor.