Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.     

The Plasticity of Molecular Interactions Governs Bacterial Microcompartment Shell Assembly

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Proton countertransport by the reconstituted erythrocyte Ca2+-translocating ATPase: Evidence using ionophoretic compounds

Summary Human erythrocyte Ca²⁺-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca²⁺ inside the vesicles, both in the absence and in the presence of the Ca²⁺-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca²⁺ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca²⁺/2H⁺ ionophore A23187 (9 to 10-fold). A Ca²⁺ ionophore unable to translocate H⁺, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca²⁺ ionophore, the electroneutral K⁺(Na⁺)/H⁺ ionophoretic exchanger, nigericin, or the electroneutral Na⁺(K⁺)/H⁺ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca²⁺-ATPase not only translocates Ca²⁺ but also H⁺ in the opposite direction.     

Congenital adrenal hypoplasia, myopathy, and glycerol kinase deficiency: Molecular genetic evidence for deletions

Glycerol kinase deficiency (GKD) is an X-linked recessive trait that occurs in association with congenital adrenal hypoplasia (AH) and developmental delay with or without congenital dystrophic myopathy. Several such patients have recently been reported to have cytological deletions of chromosome region Xp21 and/or of DNA markers that map near the locus for Duchenne muscular dystrophy (DMD) in band Xp21. We have examined the initial family reported in the literature and, using prometaphase chromosome studies and Southern blot analysis with 13 different DNA probes derived from band Xp21, have found no deletions within this region of the X chromosome. When DNA samples from six other unrelated affected males were analyzed, four of them were found to have different-size deletions within Xp21. Thus, the form of GKD associated with AH and dystrophic myopathy exhibits significant genetic heterogeneity at the DNA level. No deletions were detected in two patients with isolated GK deficiency. Comparison of our molecular studies of unrelated patients with deletions of DNA segments allows us to define the region of Xp21 (between probes J-Bir and L1.4) that most likely contains the genes for GKD and AH. This location is distal to the DMD locus. The patients with progressive muscular dystrophy tended to have larger deletions that include markers known to derive from the DMD locus, while GKD/AH/dystrophic-myopathy patients without current evidence of deletion seemed to have a milder, nonprogressive form of congenital myopathy.     

Prostaglandins E2 and I2 cause greater relaxations in pulmonary veins than in arteries of newborn lambs

Gao, Yuansheng, Haiyan Zhou, Basil O. Ibe, and J. Usha Raj.Prostaglandins E₂ andI₂ cause greater relaxations inpulmonary veins than in arteries of newborn lambs. J. Appl. Physiol. 81(6): 2534-2539, 1996.Prostaglandins E₂(PGE₂) andI₂(PGI₂) are important vasoactivemediators in pulmonary vessels. The present study was designed todetermine whether the responses of pulmonary arteries to theseprostanoids are different from those of veins in newborn lambs.Fourth-generation pulmonary arterial and venous rings without endothelium were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95%O₂-5%CO₂, 37°C), and their isometric force was measured. During contraction with endothelin-1 orU-46619 (indomethacin was present to eliminate the possible involvementof endogenous cyclooxygenase products),PGE₂,PGI₂, and carbacyclin (a stableanalogue of PGI₂) inducedgreater relaxations in veins than in arteries. In both vessel types,relaxations induced by PGE₂ weregreater than those induced by PGI₂or carbacyclin. Forskolin, an activator of adenylate cyclase, alsoinduced greater relaxation of veins than of arteries. Relaxationinduced by 8-bromoadenosine 3,5-cyclic monophosphate, ananalogue of adenosine 3,5-cyclic monophosphate (cAMP),was comparable in both vessel types. Radioimmunoassay revealed that thebasal and calcium ionophore A-23187-induced releases ofPGE₂ or 6-ketoprostaglandinF₁ (the stable breakdown product of PGI₂) were similarbetween arteries and veins. Measurement of cAMP (in the presence ofisobutylmethylxanthine) showed that PGE₂ and forskolin induced greaterincrease in cAMP in veins than in arteries. Our results demonstratethat PGE₂ andPGI₂ are more potent vasodilatorsin pulmonary veins than in arteries in newborn lambs. A difference inthe activity of adenylate cyclase may contribute to the differentialresponses to PGE₂ andPGI₂ between pulmonary arteriesand veins. Furthermore, PGE₂appears play an more important role than doesPGI₂ in modulating pulmonaryvascular tone of newborn lambs.

     

Mechanism of Benzoic Acid Uptake by Saccharomyces cerevisiae

A fast uptake of the preservative benzoic acid was observed in Saccharomyces cerevisiae, reaching saturation in about two min and then remaining constant at this level. The strong dependence of benzoic acid uptake on pH was due to the relative distribution of molecular and ionic forms in solution and not to the pH itself. The molecular form was the only one taken up by the cells. The specificity of the uptake mechanism was evidenced by the pattern of irreversible heat inactivation of the uptake system resembling protein denaturation by heat. Furthermore, the effect of temperature on the uptake was similar to that observed in enzymic reactions, whereas the kinetic data of uptake conformed to the Michaelis-Menten curve of saturation with a Km of 1.54 X 10(-2) M and Vmax of 3 X 10(-3) M/10s. The evidence presented in this paper indicates that compounds of protein nature are involved in the uptake of this preservative.     

Combinatorial Blockade of Calcineurin and CD28 Signaling Facilitates Primary and Secondary Therapeutic Gene Transfer by Adenovirus Vectors in Dystrophic (mdx) Mouse Muscles

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.     

Coumarins from Amyris balsamifera

Two new coumarins have been isolated from the aerial parts of Amyris balsamifera. On the basis of spectral and chemical data, these have been identified as (R)-(+)-6-(2′-hydroxy-3′-methyl-3′-butenyl)-7-methoxycoumarin and balsamiferone, 7-hydroxy-3,6-bis (3′-methyl-2′-butenyl)-coumarin.     

中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆

以α３２ＰｄＡＴＰ标记含ＣｆＭＮＰＶ几丁质酶基因的重组质粒为探针,在６８℃条件下对棉铃虫单粒包埋核型多角体病毒（ＨａＳＮＰＶ）进行Ｓｏｕｔｈｅｒｎ杂交,将ＨａＳＮＰＶ的几丁质酶基因分别定位在ＢａｍＨＩＥ、ＢｇｌⅡＥ、ＥｃｏＲＩＧ、ＨｉｎｄⅢＦ、ＸｂａＩＨ、ＢａｍＨＩ＋ＨｉｎｄＩＩＭ和ＢａｍＨＩ＋ＸｂａＩＨ,并以ｐＴＺ１９Ｒ为载体获得了ＸｂａＩＨ片段克隆。     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.