Biomarkers and longitudinal changes in lumbar spine degeneration and low back pain: the Johnston County Osteoarthritis Project.

OBJECTIVE: To determine if baseline biomarkers are associated with longitudinal changes in the worsening of disc space narrowing (DSN), vertebral osteophytes (OST), and low back pain (LBP). DESIGN: Paired baseline (2003-2004) and follow-up (2006-2010) lumbar spine radiographs from the Johnston County Osteoarthritis Project were graded for severity of DSN and OST. LBP severity was self-reported. Concentrations of analytes (cytokines, proteoglycans, and neuropeptides) were quantified by immunoassay. Pressure-pain threshold (PPT), a marker of sensitivity to pressure pain, was measured with a standard dolorimeter. Binary logistic regression models were used to estimate odd ratios (OR) and 95% confidence intervals (CI) of biomarker levels with DSN, OST, or LBP. Interactions were tested between biomarker levels and the number of affected lumbar spine levels or LBP. RESULTS: We included participants (n = 723) with biospecimens, PPT, and paired lumbar spine radiographic data. Baseline Lumican, a proteoglycan reflective of extracellular matrix changes, was associated with longitudinal changes in DSN worsening (OR = 3.19 [95% CI 1.22, 8.01]). Baseline brain-derived neuropathic factor, a neuropeptide, (OR = 1.80 [95% CI 1.03, 3.16]) was associated with longitudinal changes in OST worsening, which may reflect osteoclast genesis. Baseline hyaluronic acid (OR = 1.31 [95% CI 1.01, 1.71]), indicative of systemic inflammation, and PPT (OR = 1.56 [95% CI 1.02, 2.31]) were associated with longitudinal increases in LBP severity. CONCLUSION: These findings suggest that baseline biomarkers are associated with longitudinal changes occurring in structures of the lumbar spine (DSN vs OST). Markers of inflammation and perceived pressure pain sensitivity were associated with longitudinal worsening of LBP.

The Direct Assessment of Nonvertebral Fractures in Community Experience (DANCE) study: 2-year nonvertebral fragility fracture results.

UNLABELLED: This observational study evaluated the occurrence of nonvertebral fragility fractures (NVFX) in over 4,000 men and women with osteoporosis treated with teriparatide (TPTD). The incidence of new NVFX decreased for patients receiving TPTD treatment for greater than 6 months. No new significant safety findings were observed in this large trial. INTRODUCTION: The Direct Assessment of Nonvertebral Fractures in Community Experience (DANCE) study evaluated the occurrence of NVFX in patients receiving TPTD for osteoporosis in a real-world setting. METHODS: DANCE is a multicenter, prospective, observational trial that examined the long-term effectiveness of TPTD in men and women with osteoporosis whom study physicians judged to be suitable for TPTD therapy. Patients received 20 µg TPTD per day by subcutaneous injection for up to 24 months and were followed for 24 months after treatment cessation. The incidence of patients experiencing a new NVFX, defined as a fracture associated with low trauma, was evaluated during four 6-month periods in both the treatment and cessation phases with >0 to ≤6 months serving as the reference. We also observed the spectrum and occurrence of serious adverse events. RESULTS: Of the 4,167 patients enrolled, 4,085 took one or more doses of TPTD (safety population); 3,720 were included in the efficacy analysis. The incidence of patients experiencing a NVFX was 1.42, 0.91, 0.70, and 0.81 % for the four treatment periods, respectively, and 0.80, 0.68, 0.33, and 0.33 % for the four periods after treatment cessation. Differences for each period were statistically significant compared with the reference period (first 6-month interval, each p < 0.05). No new significant safety findings were observed. CONCLUSIONS: In this study, the incidence of NVFX decreased for patients receiving TPTD for all three treatment periods >6 months compared to 0 to ≤6 months, and this trend persisted throughout the cessation phase. TPTD was generally well tolerated.

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments.

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Visualization of head-head interactions in the inhibited state of smooth muscle myosin.

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer.

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.

Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.

Electron diffraction of frozen, hydrated protein crystals.

High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique. The use of frozen specimens to maintain the hydration of complex biological structures has certain advantages over previously developed liquid hydration techniques.

Equine chlamydiosis-An emerging infectious disease requiring a one health surveillance approach.

Psittacosis is a rare but potentially fatal zoonosis caused by Chlamydia psittaci, an organism that is typically associated with bird contact. However C. psittaci is capable of infecting other non-avian hosts, such as horses, sheep, cattle and goats. Stud staff and veterinarians have significant exposure to parturient animals and reproductive materials in their routine work. To investigate the zoonotic potential associated with the emergence of C. psittaci as an abortifacient agent in horses, we established a programme of joint human and animal surveillance in a sentinel horse-breeding region in Australia. This programme comprised cross-notification of equine cases to public health agencies, and active follow-up of known human contacts, including stud workers, foaling staff, veterinarians and laboratory staff. We identified no confirmed cases of acute psittacosis despite intensive surveillance and testing of heavily exposed contacts; however, further work in the area is needed.

The contribution of zinc to platelet behaviour during haemostasis and thrombosis.

Platelets are the primary cellular determinants of haemostasis and pathological thrombus formation leading to myocardial infarction and stroke. Following vascular injury or atherosclerotic plaque rupture, platelets are recruited to sites of damage and undergo activation induced by a variety of soluble and/or insoluble agonists. Platelet activation is a multi-step process culminating in the formation of thrombi, which contribute to the haemostatic process. Zinc (Zn(2+)) is acknowledged as an important signalling molecule in a diverse range of cellular systems, however there is limited understanding of the influence of Zn(2+) on platelet behaviour during thrombus formation. This review evaluates the contributions of exogenous and intracellular Zn(2+) to platelet function and assesses the potential pathophysiological implications of Zn(2+) signalling. We also provide a speculative assessment of the mechanisms by which platelets could respond to changes in extracellular and intracellular Zn(2+) concentration.

Effects of solutes on the formation of crystalline sheets of the Ca(2+)-ATPase in detergent-solubilized sarcoplasmic reticulum.

The Ca(2+)-ATPase crystals formed in detergent solubilized sarcoplasmic reticulum (SR) at 2 degrees C in a crystallization medium of 0.1 M KCl, 10 mM K-Mops (pH 6.0), 3 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 20% glycerol and 20 mM CaCl2 (J. Biol. Chem. 263, 5277 and 5287 (1988)) contain highly ordered sheets of ATPase molecules, that associate into large multilamellar stacks (greater than 100 layers). When the crystallization is performed in the same medium but in the presence of 40% glycerol at low temperature the stacking is reduced to 4-5 layers and the average diameter of the crystalline sheets is increased from less than 1 micron to 2-3 microns. Glycerol and low temperature presumably reduce stacking by interfering with the interactions between the hydrophilic headgroups of Ca(2+)-ATPase molecules in adjacent lamellae, while not affecting or promoting the ordering of ATPase molecules within the individual sheets. Electron diffraction patterns could be regularly obtained at 8 A and occasionally at 7 A resolution on crystals formed in 40% glycerol, either at 2 degrees C or at -70 degrees C. In the same media but in the absence of glycerol, polyethyleneglycol 1450, 3000 and 8000 (1-8%) induced the formation of ordered crystalline arrays containing 10-12 layers that were similar to those obtained in 40% glycerol. Replacement of 40% glycerol with 10-50% glucose or supplementation of the standard crystallization medium with polyethyleneglycol (PEG 3000 or 8000; 1, 2, 5 and 8%) had no beneficial effect on the order of crystalline arrays compared with media containing 40% glycerol.

Intragenic matrix attachment and DNA-protein interactions in the human X-linked Hprt gene.

To investigate the possible contribution of intragenic differentially methylated cytosines to X-linked gene expression, we examined DNA-protein interactions in a region in intron 3 of the human hypoxanthine phosphoribosyltransferase gene which contains at least one HpaII site methylated specifically on the active X. In vitro DNase I footprinting experiments using unmethylated DNA and HeLa nuclear extract identified three footprints (I-III). Footprints I and III flank an Alu repeat containing the HpaII site(s), one of which is contained within footprint II. Although methylation of the HpaII site had no effect on footprint II binding interactions, methylation of nearby CpGs substantially reduced the formation of three of the specific DNA-protein complexes binding to footprint II in mobility shift assays. Additionally, an A+T rich region immediately 5' to the HpaII-containing Alu repeat was found to bind specifically to nuclear matrices in vitro. We suggest that differential methylation of CpGs may affect the binding of regulatory proteins in vivo, and that interactions between the footprint proteins and those binding to the matrix attachment region may be involved in controlling X-linked Hprt expression.

Projection image of smooth muscle alpha-actinin from two-dimensional crystals formed on positively charged lipid layers.

Two-dimensional crystalline arrays of chicken gizzard alpha-actinin have been formed on positively charged lipid layers. This is the first reported crystallization of alpha-actinin. The crystals have unit cell dimensions of a = 248 A, b = 194 A, y = 106 degrees and contain two alpha-actinin molecules. The two-sided group is P21. Projection images obtained from electron micrographs of negatively stained crystals have been calculated to a resolution of 25 A. These images reveal a complex substructure. The molecule in projection is 340 A in length and has 12 density peaks that probably correspond to protein domains. A pair of peaks is found at each end of the molecule, these probably correspond to the actin binding region. Eight peaks are observed in the central, rod-shaped region, these may correspond to the spectrin-like repeats predicted from the amino acid sequence. However, these eight central peaks are not arranged in four pairs but, instead, consist of three central pairs flanked at either end by a single peak, which appears larger and denser in projection than the three central pairs. The individual alpha-actinin molecules in projection lack 2-fold symmetry suggesting that either smooth muscle alpha-actinin lacks a molecular 2-fold symmetry axis or that the molecular 2-fold is not parallel with the crystallographic 2-fold axis. The ends of the molecule have different appearance in projection, suggesting that the molecule is twisted about the long axis. A hypothesis is proposed to explain the variations in molecular length and Ca2+ sensitivity between alpha-actinin isoforms.

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum.

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.

The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding.

The three-dimensional structure of alpha-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. alpha-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpretation of the 15 A-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of alpha-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca(2+)-dependent actin binding of non-muscle isoforms.

Three-dimensional arrangement of the cell wall protein of Sulfolobus acidocaldarius.

The three-dimensional structure of the S-layer that surrounds the bacterium Sulfolobus acidocaldarius is described in detail. Pieces of the S-layer, which are two-dimensional crystals with p6 symmetry, have been studied by crystallographic analysis of electron micrographs of tilted specimens. In the density map, each asymmetric unit appears to consist of several domains connected by strong hinges. On the basis of the ragged appearance of the structure at torn edges, we now suggest that the single species of polypeptide is in a highly extended conformation, with much overlap between different molecules, and show how such molecules might be weaved together to produce the morphological domains. We show that the closed surface lattice of the intact cell wall contains 5-fold and 7-fold vertices and show how the subunit structure appears to be well suited to form 5-fold and 7-fold symmetric rings at these points in place of the 6-fold rings of the hexagonal lattice.

Crossbridges in the complete unit cell of rigor insect flight muscle imaged by three-dimensional reconstruction from oblique sections.

We have computed two types of 3-D reconstructions from single images of oblique transverse sections through rigor insect flight muscle (IFM) that permit simultaneous examination of all myosin crossbridges within the unit cell. One type, crystallographic serial section reconstruction (CSSR), utilizes primarily real space image manipulations of the periodic crossbridge lattice to obtain a 3-D reconstruction from a single image. The CSSRs, which do not average successive unit cells along the filament axis, reveal variations in the rigor double chevrons within the 116 nm long axial repeat and in particular show that specific crossbridges are absent. CSSRs establish that in rigor, the 116 nm period contains nine 12.9 nm repeats of attached crossbridges rather than the eight 14.5 nm repeats of myosin head origins observed in the relaxed state. This indicates that dominance of the actin repeat on myosin head form enforces axial and azimuthal changes on the crossbridge origins on the thick filament. The second type, superlattice reconstruction (SLR), is carried out entirely in Fourier space and produces an averaged reconstruction with the symmetry of the unit cell enforced. SLRs measure the 3-D transform of the complete unit cell, permitting direct comparison with X-ray diagrams from native muscle. Averaging several SLRs together has produced the highest resolution reconstruction of IFM to date. Oblique section reconstructions made by both methods confirm in greater detail the presence of two-headed lead crossbridges and single-headed rear crossbridges implying heads with differing angles and conformation. Reduced twist in the thin filament coincident with the lead crossbridge is also apparent. We have modeled several interpretations of this reduced twist in terms of structural changes in both myosin and actin at the lead bridge. In addition, these 3-D images resolve a feature just Z-ward of the rear crossbridge where antibody labeling has identified part of the large troponin complex of IFM.

Electron tomography of insect flight muscle in rigor and AMPPNP at 23 degrees C.

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed "lead" bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar comparison using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.

Three-dimensional structure of nucleotide-bearing crossbridges in situ: oblique section reconstruction of insect flight muscle in AMPPNP at 23 degrees C.

We have explored the three-dimensional structure of myosin crossbridges in situ in order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by approximately 2.0 nm and tension is reduced by approximately 70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimensional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP "lead" crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in situ rigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.

Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice to Trypanosoma congolense infection.

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.