High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

Background

Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.

Methods

A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP^® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.

Results

The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R² = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).

Conclusions

High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.     

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b₅ and NADH-cytochrome b₅ reductase from the microsomal fraction of Pisum sativum. The cytochrome b₅ component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b₅ component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.     

A change of osteocalcin (OC) and tartrate resistant acid phosphatase 5b (TRACP-5b) with the menstrual cycle

Bone metabolism markers associated with 4 menstrual cycle phases were evaluated in 14 healthy young females without menstrual disorder. Menstrual cycle phases were confirmed with basal body temperature for 3 months, luteinizing hormone kits, and sexual hormone concentrations of serum. The bone metabolism markers used were osteocalcin (OC), which was measured by immunoradiometric assay (IRMA), and tartrate resistant acid phosphatase 5b (TRACP-5b), which was measured by enzyme immunometric assay (EIA). The highest values of OC and TRACP-5b were observed in the ovulation phase, and TRACP-5b increased significantly when compared with levels in the menstrual phase (p<0.05). Furthermore, the changes in sex-hormone secretion involved in OC and TRACP-5b showed specific patterns during the menstrual cycle. In other words, TRACP-5b levels are influenced by sex hormones produced during the menstrual period and are based on the bone-formation status. Therefore, it is presumed that the TRACP-5b levels during ovulation play a central role in bone formation and bone metabolism.     

Cytochrome b(5) forms homomeric complexes in living cells

Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Carbon monoxide complex of cytochrome b(5) at acidic pH

CO complex of cyt b(5) generated at acidic pH is investigated by absorption, resonance Raman (RR), and far UV CD measurements. The Soret maximum wavelength blue-shifted to 420 nm with other absorption bands observed around 540 and 570 nm for reduced cyt b(5) upon interaction with CO at acidic pH (pH 3.1-3.5). Under this condition, the iron-carbon stretching RR band was observed at 529 cm(-1) (520 cm(-1) for C(18)O), which indicated formation of a heme&bond;CO adduct with a histidine as an axial ligand. Heme dissociated from the reduced cyt b(5) protein at pH approximately 3.5, whereas its rate decreased under CO atmosphere compared with N(2) atmosphere, due to formation of a heme&bond;CO adduct with a histidine as an axial ligand.     

Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a)

Receptor tyrosine kinases activate mitogen-activated protein (MAP) kinases through Ras, Raf-1, and MEK. Receptor tyrosine kinases can be transactivated by G protein-coupled receptors coupling to G(i) and G(q). The human G protein-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) couple to G(s) and elevate intracellular cAMP. Certain G(s)-coupled receptors have been shown to activate MAP kinases through a protein kinase A- and Rap1-dependent pathway. We report the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 (p44 and p42 MAP kinase) through the human serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in COS-7 and human embryonic kidney HEK293 cells. In transfected HEK293 cells, 5-HT-induced activation of ERK1/2 is sensitive to H89, which indicates a role for protein kinase A. The observed activation of ERK1/2 does not require transactivation of epidermal growth factor receptors. Furthermore, 5-HT induced activation of both Ras and Rap1. Whereas the presence of Rap1GAP1 did not influence the 5-HT-mediated activation of ERK1/2, the activation of ERK1/2 was abolished in the presence of dominant negative Ras (RasN17). ERK1/2 activation was reduced in the presence of "dominant negative" Raf1 (RafS621A) and slightly reduced by dominant negative B-Raf, indicating the involvement of one or more Raf isoforms. These findings suggest that activation of ERK1/2 through the human G(s)-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in HEK293 cells is dependent on Ras, but independent of Rap1.     

Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5)

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.     

The N-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.     

Poly(ε-L-lysine): Synthesis and conformation

The synthesis of poly(ε-L -lysine) is described. This is a poly(ε-amino acid) in which the ε-amino group of lysine is condensed with the α-carboxyl group to produce a chain backbone that is a variant of the usual one seen in proteins and the side chain is the α-amino group. Conformational studies of poly(ε-L -lysine) and its t-butyloxycarbonyl derivative suggest the likelihood of a chain order that is formally similar to the antiparallel pleated-sheet conformation of proteins.     

Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5)

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b5 as a GST-cyt b5 fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein.     

Characterization and biosynthesis of cytochrome b(5) in rat liver microsomes

1. Cytochrome b₅ is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b₅ was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide `fingerprint' patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b₅ after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b₅ under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome.     

Synaptic organization of the cat parietal association cortex (area 5b)

An electron microscopy study was made of synaptic organization in the cat association cortex, area 5b. A total of 1635 axonal terminals were discovered over 6215 µm² (240 electronic imagings of slices of different association cortex layers); i.e., an average of 263±16 terminals per 1000 µm² expanse. It was found that 75.5% of axon terminals contained synaptic vesicles and formed either one- or two-sided contact with postsynaptic structures; 24.5% of axonal terminals contained synaptic vesicles but formed no distinct synaptic contacts with nearby neurons; 84.9% of terminals contained round-shaped or slightly oval synaptic vesicles; 7.8% had both rounded and elongated shapes, and vesicles were very elongated in the remaining 7.3%. Of the axonal terminals having synaptic contacts, axo(dendritic)-spinal terminals accounted for 46.6%, and axodendritic and axosomatic endings amounted to 50.0% and 3.4% respectively (in all 77% of axosomatic terminals contained elongated vesicles and maintained symmetrical contact, while 23% had round-shaped vesicles and formed asymmetrical contact). Calculations show that for each 1 mm³ an average of 258 million axonal terminals are found forming synaptic contacts in the cat association cortex as well as 84 million terminals containing synaptic vesicles but not forming contact.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 174–185, March–April, 1989.     

Homeobox b5 (Hoxb5) regulates the expression of Forkhead box D3 gene (Foxd3) in neural crest

Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).     

OnpA, an unusual flavin-dependent monooxygenase containing a cytochrome b(5) domain

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b(5) domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b(5) domain were associated with binding of the heme, and the cytochrome b(5) domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b(5) domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b(5) and flavin-dependent monooxygenases.     

NADH-细胞色素 b5 还原酶在甲状腺过氧化氢生物合成中的作用

应用高香草酸荧光分析技术及NADH－高铁氰化钾还原酶法,对正常和Graves病甲状腺过氧化氢（H2O2）和NADH－细胞色素b5还原酶（b5R)进行测定,发现Graves病甲状腺b5R活性和H2O2水平均明显高于正常,而H2O2酶活性在Graves病和正常甲状腺间无显著差异。加b5R抑制剂对氯汞苯甲酸抑制b5R活性,Graves病和正常甲状腺b5R活性降低近85％,同时H2O2降低近50％,蛋白结合碘形成减少近52％。b5R活性和H2O2水平两者呈显著正相关关系。以上结果表明,b5R参与甲状腺内H2O2的生物合成,是甲状腺内产生H2O2的重要酶系。