Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

Ornithine transcarbamylase (OTC) deficiency in a female patient with a de novo deletion of the paternal X chromosome

Summary A girl with ornithine transcarbamylase (OTC) deficiency was investigated for molecular and cytogenetic abnormalities that might explain this phenotype. Analysis with polymorphic DNA markers indicated that the patient did not inherit paternal alleles of the OTC locus, but that she did inherit the proximal locus DXS7 and the long arm of chromosome X. High-resolution cytogenetic analysis of the patient indicated a deletion of Xp11.4-p21, whereas both parents had normal karytoypes. Since the mother might be heterozygous according to biochemical tests, a second mutation within the maternal OTC gene cannot be excluded.     

Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database. Modification of the large subunit depends on cellular proliferation

Nuclear Factor IV (NFIV) is a heterodimeric DNA-binding protein from HeLa cells, recognizing molecular ends and is identical to the autoantigenic target Ku. We have identified the two NFIV/Ku subunits, by comigration, in the 2D-gel database of transformed human amnion cell (AMA) proteins. We observed that the large subunit of NFIV/Ku consists of at least 3 charge variants that correspond to SSP IEFs 5705 (81.2 kDa, pI 5.74), 6707 (81.2 kDa, pI 5.67) and 6706 (81.9 kDa, pI 5.60) in the AMA catalogue. The relative amounts of the 2 major variants (IEFs 5705 and 6707) was dependent on the state of cell proliferation. Inhibition of DNA-synthesis by hydroxyurea also changed the relative levels of the variants, whereas aphidicolin or a thymidine block had no effect. These results suggest a possible role for NFIV/Ku in DNA replication.     

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.