Interaction of bovine ceruloplasmin with erythrocytes

Binding of ceruloplasmin to bovine erythrocytes was investigated. The interaction was specific as demonstrated by more than 85% inhibition by excess ceruloplasmin protein. Binding was also inhibited by copper-nitrilotriacetate. The KD for both intact erythrocytes and solubilized ghost membranes was of the order of 10(-7) M. Using an affinity column a specific ceruloplasmin-binding protein was isolated from ghost membranes. The protein has an apparent molecular mass of 100,000 with subunits of 45 and 50 KDa.     

Thiol-linked peroxidase activity of human ceruloplasmin

Human ceruloplasmin exhibited different antioxidant effects according to the electron donors in a metal-catalyzed oxidation system. Purified ceruloplasmin did not play a significant role in the protection of DNA strand breaks in the ascorbate/Fe3+/O2 system. However, when ascorbates were replaced with a thiol-reducing equivalent such as dithiothreitol, DNA strand breaks were significantly prevented by the same amount of ceruloplasmin. Ceruloplasmin did not catalyze the decomposition of H2O2 in the absence of reduced glutathione. On the contrary, ceruloplasmin showed a potent peroxidase ability to destroy H2O2 in the presence of reduced glutathione. In conclusion, the removal of H2O2 by human ceruloplasmin is not simply stoichiometric but thiol-dependent.     

Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Effect of ceruloplasmin on 6-hydroxydopamine oxidation

The effects of ceruloplasmin, a blu copper-containing serum glycoprotein, on the oxidation of the neurotoxin 6-hydroxydopamine in several chemical environments were studied. The spontaneous autoxidation of 6-hydroxydopamine proceeded by a free radical chain reaction involving O2-. and produced the corresponding chromogen 6-hydroxydopaminequinone and hydrogen peroxide. The process was accelerated in the presence of horse plasma ceruloplasmin. Yields of hydrogen peroxide in the presence of ceruloplasmin were significantly less than those measured in its absence. This is the first evidence of oxidase activity of ceruloplasmin toward a natural substrate, the 6-hydroxydopamine.     

Serum ceruloplasmin as a diagnostic marker of cancer

Immunohistochemical staining of MMP-9 and the type-IV collagen was performed on paraffin sections of endometrial carcinoma. Immunostaining in 129 cases of endometrial cancer detected MMP-9 in 19.0% of the cases. MMP-9 positive was shown in 30% of the cases with vessel invasion, and in 12.7% of the cases without vessel invasion (p < 0.05). MMP-9 showed positive in many cases with poor differentiation and lymph node metastasis, but still failed to achieve statistical significance. MMP-9 staining did not correlate with disease outcomes. We can not clarify that MMP-9 is associated with tumor-cell invasion and metastasis. Type-IV collagen deposition at the tumor-stromal border was studied in 58 cases of endometrial carcinoma in which disruptions were seen in varying degrees. The type-IV collagen in the primary lesion decreased as the differentiation decreased. Even in the lymph node metastasis lesions, the type-IV collagen was stained and was almost in agreement with the primary lesions. In the primary lesions, there was no relationship between MMP-9 staining and the type-IV collagen. It was suggested that the type-IV collagen observed in endometrial carcinoma was more concerned with the differentiation of the tumor than with the degradation by MMP-9.     

Zinc, copper and ceruloplasmin in arteriosclerosis

High serum levels of Zn, Cu and Ceruloplasmin were found in 13 patients with arteriosclerosis when compared with controls. The Zn/Cu ratio is much higher in arteriosclerotic patients than in healthy subjects.     

Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.     

Intracellular alpha-amylase of Streptococcus mutans

Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.     

Materials for performing external quality control of the measurement of ceruloplasmin activity

Quaternary monophosphonium compound 1-triphenyl phosphoniomethyl naphthalene bromide stabilizes blood serum samples and preserves the activity of ceruloplasmin (CP 1.16.3.1) intact for 30 days. For preparing the reference material, 10 ml of serum is added to 190 ml of 1.55 mM solution of this compound, left for 24 h at 4-8 degrees C, poured in small flasks, stored at the same temperature, and used as reference material as needed for checking up the correctness of ceruloplasmin measurements in biochemical laboratories.     

Isolation of nonlabile human ceruloplasmin by chromatographic removal of a plasma metalloproteinase

Ceruloplasmin (EC 1.16.3.1) is a copper-containing alpha 2-glycoprotein and a member of the acute phase reactant family. Fragmentation of ceruloplasmin during purification and storage has hampered studies of its structure, but it has been shown to be a 132-kDa monomer. Combining two published chromatographic steps with additional gel filtration and fast protein liquid chromatography (FPLC) steps, we now report a procedure that yields a highly purified and nonlabile protein. Human plasma was subjected to QAE-Sephadex A-50 chromatography, precipitated with ammonium sulfate, and chromatographed on a hydroxyapatite column. The resulting protein was > 95% pure but highly unstable as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; at 37 degrees C the 132-kDa protein disappeared completely within 48 h. Further purification of ceruloplasmin by Sephadex G-50 chromatography and Mono Q FPLC yielded a protein that was essentially pure by multiple criteria and completely stable even after incubation at 37 degrees C for 4 weeks. When purified ceruloplasmin was reconstituted with fractions eluted from the Sephadex G-50 column, a single fraction induced proteolytic degradation. The degradation of ceruloplasmin by this fraction was inhibited by EDTA and 1,10-phenanthroline, indicating that a plasma metalloproteinase is responsible for degradation of ceruloplasmin.     

Intracellular bacteria in protozoa

Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.     

Diagnostic value of ceruloplasmin, haptoglobin and sialic acid in chronic pyelonephritis

The widespread use of organophosphate pesticides creates the possibility of excessive exposure of migrant farm workers to these compounds. Blood cholinesterase determinations were used to compare the organophosphate pesticide exposure of 57 migrant farm workers with that of 35 controls. Frequently reported symptoms of the farm workers which might be related to pesticide exposure were also studied, including headaches, dizziness, loss of weight, nausea, and a general feeling of weakness or loss of energy. Significantly depressed cholinesterase activities were found in the farm workers, with 10.5% of the farm workers having values below the lower limit of normal. There was no significant relationship between frequently reported symptoms of the farm workers and depressed cholinesterase levels.     

Comparative antioxidant and cardioprotective effects of ceruloplasmin, superoxide dismutase and albumin

The antioxidant effects of ceruloplasmin (CAS 9031-37-2) against oxygen free radicals (.O2-, .OH, 1O2) and their by-products (H2O2, HOCl), generated by electrolysis of Krebs-Henseleit buffer, were determined in vitro by the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method and ex vivo by quantifying cardiodynamic variables of the isolated perfused rat heart. Purified ceruloplasmin (1 mumol/l) displayed a high antioxidant capacity in vitro (89.2%), while the scavenging capacity of superoxide dismutase (SOD) in equimolar concentrations was 38.1%. A relatively high scavenging activity (72.1%) was observed with bovine serum albumin (BSA). A control group of Langendorff isolated rat hearts (n = 8) was submitted to electrolysis (10 mA, for 1 min) without treatment, whereas the treated groups were perfused with ceruloplasmin, SOD or BSA (1 mumol/l) in the inflow cannula for 5 min before, during, and 5 min after electrolysis. The cardioprotective effect afforded by ceruloplasmin (83-89%) was higher than that observed with the same optimal dose of 1 mumol/l SOD (20-45%). With BSA, no protection was observed ex vivo. Particularities in scavenging specificities and mechanisms seem to explain the important differences between in vitro and ex vivo antioxidant capacities for these proteins.     

Intracellular retention of recombinant GABAB receptors

gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.     

Screening for Wilson's disease in patients with liver diseases by serum ceruloplasmin

BACKGROUND/AIMS: A low serum ceruloplasmin level is considered a diagnostic test for Wilson's disease. To examine whether it is useful to detect presymptomatic patients with Wilson's disease, serum ceruloplasmin was determined by radial immunodiffusion (normal: 20-60 mg/dl) in all patients (n = 2867) admitted for evaluation of a liver disease in 1993 and 1994. METHODS: Patients with levels lower than 20 mg/dl were further evaluated by determination of serum copper concentration, urine copper excretion and ophthalmological examination. If possible, a liver biopsy was performed and the hepatic copper content was determined by flame atomic absorption spectroscopy. RESULTS: Seventeen patients had serum ceruloplasmin levels < 20 mg/dl. One had asymptomatic Wilson's disease (no Kayser-Fleischer rings or neurological symptoms). In the other 16 patients Wilson's disease was excluded. Based on elevated hepatic copper concentration, there were considered as heterozygous carriers of the WD gene. The remaining patients had various liver diseases (acute viral hepatitis in three, chronic hepatitis in two, drug-induced liver disease in three, alcoholic induced liver disease in two) or malabsorption (n = 3). CONCLUSIONS: The positive predictive value of low serum ceruloplasmin was only 5.9%. Although helpful for identifying presymptomatic Wilson's disease, screening by determination of serum ceruloplasmin in unselected patients with clinical or laboratory evidence of liver disease is neither feasible nor cost effective.