Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

Intracellular distribution of DNA methyltransferase during the cell cycle

The intracellular distribution of DNA methyltransferase has been analyzed in synchronously proliferating human cells. The localization of DNA methyltransferase was determined immunocytochemically using monoclonal antibodies directed against this enzyme. DNA methyltransferase was found to accumulate predominantly in nuclei with weak cytoplasmic staining. The DNA methyltransferase antigen was absent in early G1 phase, appeared in late G1 prior to the onset of DNA synthesis and persisted throughout S and G2 phases of the cell cycle. Mitotic cells showed a particularly strong staining intensity. These results show that DNA methyltransferase levels fluctuate during the cell cycle. This has possible implications on the stability of the DNA methylation pattern.     

Distribution of isoaccepting tRNAs and codons for proline and glycine in collagenous and noncollagenous chicken tissues

The relation between codon usage and tRNA content for proline and glycine, the major constituents of collagen, was studied in two tissues: the magnum of laying hen oviduct and the leg tendons of chick embryo where collagen is produced. Although the relative contents of tRNA(GCCGly) and tRNA(IGGPro) in tendons, as compared to magnum indicate a specialization of the tRNA population for collagen synthesis, the distribution of the preponderant codons in collagen mRNA is correlated but at a lesser extent to that of their cognate tRNAs.     

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAsGly and iso-tRNAsPro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.     

A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor

The generation of two cleavage products of human C3, termed C3o and C3p, by incubation with a C3-cleaving protease isolated from cobra venom (Naja naja siamensis) is described. The venom protease removes the C3p fragment (Mr approximately 33,000) from the C3dg region of the C3 alpha-chain. The major cleavage fragment C3o (Mr approximately 140,000) contains the unaltered beta-chain of C3 and two alpha-chain-derived polypeptides of Mr approximately 29,000 and Mr approximately 38,000, respectively. Amino-terminal amino acids sequence analysis of C3p and the three chains of C3o allowed the identification of the exact location of the two alpha-chain-derived fragments of C3o and the three cleavage sites of the venom protease. The chain structure of C3o resembles those of C3c and cobra venom factor. In contrast to C3c but like cobra venom factor (and C3b), C3o was found to support the activation of the serine protease Factor B by cleavage in the presence of Factor D and Mg2+ into Bb and Ba, generating an enzymatically active complex that is able to cleave a fluorogenic peptide substrate for C3 convertases. Since the only stretch of amino acid residues of C3o not present in C3c is the carboxyl terminus of the Mr approximately 29,000 chain of C3o, it is suggested that this region is important for the interaction with Factor B and convertase formation.     

Methylmercury effects on cell cycle kinetics

Methylmercury (MeHg) effects on cell cycle kinetics were investigated to help identify its mechanisms of action. Flow cytometric analysis of normal human fibroblasts grown in vitro in the presence of BrdU allowed quantitation of the proportion of cells in G1, S, G2 and the next G1 phase. This technique provides a rapid and easily performed method of characterizing phase lengths and transition rates for the complete cell cycle. After first exposure to MeHg the cell cycle time was lengthened due to a prolonged G1. At 3 microM MeHg the G1 phase length was 25% longer than the control. The G1/S transition rate was also decreased in a dose-related manner. Confluent cells exposed to MeHg and replated with MeHg respond in the same way as cells which have not been exposed to MeHg before replating. Cells exposed for long times to MeHg lost a detectable G1 effect, and instead showed an increase in the G2 percentage, which was directly related to MeHg concentration and length of exposure. After 8 days at 5 microM MeHg, 45% of the population was in G2. The G2 accumulation was reversible up to 3 days, but at 6 days the cells remained in G2 when the MeHg was removed. Cell counts and viability indicated that there was not a selective loss of cells from the MeHg. MeHg has multiple effects on the cell cycle which include a lengthened G1 and decreased transition probability after short term exposure of cycling cells, and a G2 accumulation after a longer term exposure. There were no detectable S phase effects. It appears that mitosis (the G2 accumulation) and probably synthesis of some macromolecules in G1 (the lengthened G1 and lowered transition probability) are particularly susceptible to MeHg.     

Cementum annulation and age determination in Homo sapiens. II. Estimates and accuracy

The cementum annulation aging technique was evaluated in a sample of 80 clinically extracted premolars (age range 11–70 years). Demineralized thin sections (7μm) stained with hematoxylin were used. The correlation (r) between age and adjusted count (number of annulations added to age of tooth eruption) was 0.78 for the entire sample (N = 73) and 0.86 for a subsample in which teeth with periodontal disease were excluded (N = 55). Standard error of the estimates ranged from 4.7 to 9.7 years depending on sex and health status of the tooth. The technique provided significantly better estimates for females than for males. The overall inaccuracy (mean absolute error) of the technique was 6.0 years, with a bias (mean error) of 0.26 years. Reduced major axis regression of adjusted count on age produced a slope of 0.797 for the entire sample and 0.889 for the nonperiodontal disease subsample. These slopes are consistent with a hypothesis of annual deposition of cementum rings given a decrease in cementogenesis with increasing age.     

Effect of covalent attachment of immunoglobulin fragments on liposomal integrity

Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.     

Monoclonal antibody-mediated inhibition of interferon-gamma-induced macrophage antiviral resistance and surface antigen expression

A monoclonal antibody (MAb) with specificity for murine interferon-gamma (IFN-gamma) was used as a probe for studying the effect of recombinant IFN-gamma (rIFN-gamma) on antiviral activity, Fc receptor expression, and Ia antigen induction in macrophages. Cultures of C3H/HeJ peritoneal exudate macrophages were used to allow direct comparison of all three functions in the same target cell system. Our data provide two major findings: the efficacy of the MAb is very different depending on whether murine fibroblasts or macrophages are used as the target cell in the antiviral assay, i.e., greater than 20 to 100 times more MAb was required to block antiviral activity in macrophage cultures; and 10 to 50 times more MAb was required to inhibit Fc receptor vs Ia antigen expression in response to rIFN-gamma. These latter findings confirm and extend previous observations, which indicate that the induction pathways of two important differentiation markers by IFN-gamma may be dissociable.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.