Liquid chromatographic assay for a butenolide endothelin antagonist (PD 156707) in plasma

A sensitive and selective liquid chromatographic assay for determining the non-peptide endothelin A receptor antagonist PD 156707 (I) in rat plasma has been developed and validated. The analyte was isolated from matrix by solid-phase extraction. Liquid chromatographic separation was achieved isocratically ona 3.2 mm I.D., ODS column with a mobile hase of acetonitrile-ammonium phosphate (50 mM, pH 3.5) (44:56, v/v). Column effluent was monitored fluorometrically. Peak-height ratios (analyte/IS) were proportional to I concentrations in rat plasma from 25 to 1000 ng/ml. Assay precision and accuracy for I, based on quality controls, was 9.5% relative standard deviation, with relative error of ±6.5%. The quantitation limit was 25 ng/ml for a 200-μl sample aliquot.     

An experimental method for measuring force on the spinal facet joint: description and application of the method.

A technique is described for measuring load magnitude and resultant load contact location in the facet joint in response to applied loads and moments, and the technique applied to the canine lumbar spine motion segment. Due to the cantilever beam geometry of the cranial articular process, facet joint loads result in surface strains on the lateral aspect of the cranial articular process. Strains were quantified by four strain gages cemented to the bony surface of the process. Strain measured at any one gage depended on the loading site on the articular surface of the caudal facet and on the magnitude of the facet load. Determination of facet loads during in vitro motion segment testing required calibration of the strains to known loads of various magnitudes applied to multiple sites on the caudal facet. The technique is described in detail, including placement of the strain gages. There is good repeatability of strains to applied facet loads and the strains appear independent of load distribution area. Error in the technique depends on the location of the applied facet loads, but is only significant in nonphysiologic locations. The technique was validated by two independent methods in axial torsion. Application of the technique to five in vitro canine L2-3 motion segments testing resulted in facet loads (in newtons, N) of 74+ / -23 N (mean + / -STD) in 2 newton-meter, Nm, extension, to unloaded in flexion. Lateral bending resulted in loads in the right facet of 40+ / -32 N for 1 Nm right lateral bending and 54+ / -29 N for 1 Nm left lateral bending. 4 Nm Torsion with and without 100 N axial compression resulted in facet loads of 92+ / -27 N and 69+ / -19 N, respectively. The technique is applicable to dynamic and in vivo studies.     

Area and the latitudinal diversity gradient for terrestrial birds

We tested the hypothesis that area represents the primary explanation for the latitudinal diversity gradient using breeding terrestrial birds of North America, the northern Palearctic, Australia and the Afrotropics as our focal group. We tested two propositions inherent to the area hypothesis: (1) tropical biomes are larger than extra-tropical biomes, and (2) there is a significant species-area relationship for birds at the biome scale of resolution. Using a more realistic definition of biomes which incorporates the effects of both energy and water, we find no support for either proposition, leading us to conclude that the area per se does not explain terrestrial latitudinal diversity gradients.     

Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

Maspin binds to urokinase-type and tissue-type plasminogen activator through exosite-exosite interactions

Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K(d) values between 300 and 600 nM. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg(340) within the reactive center loop. The binding site on tPA does not involve the proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the proteinase.     

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.     

Endo-[beta]-Mannanase Activity from Individual Tomato Endosperm Caps and Radicle Tips in Relation to Germination Rates

Endo-[beta]-mannanase is hypothesized to be a rate-limiting enzyme in endosperm weakening, which is a prerequisite for radicle emergence from tomato (Lycopersicon esculentum Mill.) seeds. Using a sensitive, single-seed assay, we have measured mannanase activity diffusing from excised tomato endosperm caps following treatments that alter the rate or percentage of radicle emergence. Most striking was the 100- to more than 10,000-fold range of mannanase activity detected among individual seeds of highly inbred tomato lines, which would not be detected in pooled samples. In some cases a threshold-type relationship between mannanase activity and radicle emergence was observed. However, when radicle emergence was delayed or prevented by osmoticum or abscisic acid, the initial increase in mannanase activity was unaffected or even enhanced. Partially dormant seed lots displayed a bimodal distribution of activity, with low activity apparently associated with dormant seeds in the population. Gibberellin- and abscisic acid-deficient mutant seeds exhibited a wide range of mannanase activity, consistent with their variation in hormonal sensitivity. Although the presence of mannanase activity in the endosperm cap is consistently associated with radicle emergence, it is not the sole or limiting factor under all conditions.     

Glucocorticoids in muscular dystrophy: beneficial effects of dexamethasone on avian myopathy

A corticosteroid with mixed glucocorticoid-mineralocorticoid actions was previously shown to improve neuromuscular function in muscular dystrophic chickens. The significance of that finding was recently underscored by reports that a mixed-action corticosteroid improved muscle function in Duchenne dystrophy patients, albeit at high doses. In the present study a pure glucocorticoid improved function and retarded muscle histopathology in the chicken, but a pure mineralocorticoid did not. These observations suggest that elucidation of mechanisms by which glucocorticoids beneficially affect dystrophic muscle could lead to development of more effective therapies.