Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b)

A synthetic pentapeptide analogous to an inhibitory factor associated with human granulocytes was tested in vivo on female C3H mice. The relative and absolute numbers of myelopoietic and erythropoietic cells in the bone marrow were measured following injections as well as the continuous infusion of the pentapeptide in dose ranges between 10(-8)M and 10(-4)M (0.12 micrograms to 1.2 mg/mouse). In low doses, the pentapeptide reduced the number of myelopoietic cells in the bone marrow, and this was accompanied by reduced numbers of granulocytes and monocytes and peripheral blood. Elevated doses also decreased erythropoiesis. In contrast, continuous infusion of 14 micrograms/h for 19 days seemed to make the myelopoietic cells refractory to further action. A regulatory function of the pentapeptide is proposed.     

Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b)

Abstract. Degeneration of the archenteron in middle gastrulae occurred in the presence of α,α'-dipyridyl or Zn²⁺, inhibitors of prolyl hydroxylase. In the presence of these substances the archenteron degenerated and was eventually destroyed. Adding Fe²⁺ to the embryo culture containing α,α'-dipyridyl protected the archenteron from further degeneration, but the collapsed archenteron was not restored to the upright position. At the late gastrula stage, α,α'-dipyridyl did not cause the degeneration of the archenteron. Treatment of the embryos by α,α'-dipyridyl, starting at the swimming blastula stage, resulted in the production of many mesenchyme-like cells but archenteron was not produced in the embryos. Addition of Fe²⁺ to α,α'-dipyridyl culture, just before the beginning of gastrulation of normal embryos, resulted in the formation of normal archenteron. α,α'-Dipyridyl inhibited hydroxylation of proline residues of collagen in sea urchin embryos and Fe²⁺ prevented the inhibition by α,α'-dipyridyl. Respiration was not inhibited by α,α'-dipyridyl.     

Modification of mouse hemopoietic cell proliferation in vivo by a hemoregulatory pentapeptide (HP 5b). Relation to circadian rhythms

The effects on murine hemopoietic cell proliferation of hemoregulatory peptide 5b, a synthetic factor which inhibits granulopoiesis, have been studied in vivo by 3H-thymidine labeling, cell cycle analyses by flow cytometry and total cell counts per femur. Single injections of the peptide seemed to obliterate the normal circadian rhythm of bone marrow cell proliferation. By autoradiography, the main effect was an inhibition of myelopoietic cell proliferation, although erythropoiesis was also affected. This occurred in two ways: a) an immediate effect observed in the first 24 h after a single injection, and b) a delayed effect observed 6-12 days after a week of peptide infusion which could be a result of stem cell inhibition. A secondary accumulation of cells with G2 phase DNA content was seen regularly. Thus the hemoregulatory peptide seems to influence hemopoietic cell proliferation and has its main effect on myelopoiesis. However, inhibition is less marked than observed previously in stem cells.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

The sequence of the hemoregulatory peptide is present in Gi alpha proteins

The hemoregulatory peptide PyroGlu-Glu-Asp-Cys-Lys (HP5b), which inhibits myelopoietic colony formation in vitro, is shown to be a sequence motif which is also part of the effector domain of Gi alpha proteins. Out of 8 synthetic peptides with sequence variations of HP5b, those with the closest similarity to the Gi alpha sequence are biologically active. The inhibitory effect appears to be dependent on the blocked N-terminus. It is postulated that these peptides may interfere with signal transduction mediated by Gi alpha proteins.     

Insulinotropic and gastrin-releasing action of gastrin-releasing peptide (GRP)

The effect of intravenous administration of gastrin-releasing peptide ( GRP ) on serum gastrin and insulin levels was studied in ad libitum fed and 24-h fasted rats. Administration of GRP (55 micrograms/kg body weight) caused a significant (P less than 0.05) elevation in serum gastrin levels at 10, 30, 60, and 120 min in the rats fed ad libitum, whereas in the fasted rats, gastrin levels rose significantly only at 10 min. GRP did not cause insulin release in fasted rats, but in the fed rats, it led to a significant elevation in serum insulin levels at 10 and 30 min, in comparison to controls. GRP appears to have an insulinotropic action in addition to a gastrin-releasing effect.     

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.     

果蝇胰岛素样肽8(Dilp8)的功能及作用机制

果蝇Drosophila作为一种模式生物,具有生长周期短、繁殖能力强和研究成本低等优点。而且果蝇有65%的基因与人类同源,特别是其遗传背景简单的特点,使其在生物生长发育研究、病理机制研究和基因表达调控等研究中发挥重要作用。目前在果蝇中已发现8种胰岛素样肽,即果蝇胰岛素样肽1-8(Drosophila insulin-like peptide 1-8, Dilp1-8),而对于果蝇胰岛素信号通路的研究主要集中在其调控机体生长发育和能量代谢的方面,这些功能主要通过Dilp1-7来发挥作用。对于Dilp8的功能及其发挥作用的分子机制知之甚少。本文总结了自Dilp8被发现以来,人们对于其功能的研究结果。Dilp8主要在幼虫成虫盘和成年雌果蝇的卵巢中表达,其主要作用是调节果蝇的组织生长和发育时间,使果蝇生长为具有相对固定体型和一定对称性的个体。当果蝇幼虫在生长过程中受到损伤时,Dilp8会通过延缓发育时间来缓解异常生长。Dilp8被激活后,在中枢神经系统与其受体富含亮氨酸重复序列的G蛋白偶联受体3(leucine-rich repeat-containing G protein-coupled ...     

Antimicrobial peptides (AMPs): peptide structure and mode of action

Antimicrobial peptides (AMPs) have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of AMP activity, their relevance to resolving how peptides damage and kill microorganisms still needs to be clarified. Moreover, many AMPs employ sophisticated and dynamic mechanisms of action to carry out their likely roles in antimicrobial host defense. Recently, it has been speculated that transmembrane pore formation is not the only mechanism of microbial killing by AMPs. In fact, several observations suggest that translocated AMPs can alter cytoplasmic membrane septum formation, reduce cell-wall, nucleic acid, and protein synthesis, and inhibit enzymatic activity. In this review, we present the structures of several AMPs as well as models of how AMPs induce pore formation. AMPs have received special attention as a possible alternative way to combat antibiotic-resistant bacterial strains. It may be possible to design synthetic AMPs with enhanced activity for microbial cells, especially those with antibiotic resistance, as well as synergistic effects with conventional antibiotic agents that lack cytotoxic or hemolytic activity.     

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.     

Mechanism of action of the tri-hybrid antimicrobial peptide LHP7 from lactoferricin,HP and plectasin on Staphylococcus aureus

The tri-hybrid peptide-LHP7 has the potent activity against Gram-positive and Gram-negative as well as fungi, but its mechanism of action has remained elusive. The effluences of LHP7 on the Staphylococcus aureus cell membrane and targets of intracellular action were investigated. LHP7 exhibited an inhibitory effect on the S. aureus growth, similar to those achieved by plectasin, vancomycin and gramicidin. The membrane integrity studies confirmed that LHP7 disrupted the cell membrane, indicating a membrane permeabilizing killing action. A marginal decline in the intensity fluorescence indicated no significant depolarization of the membrane potential following LHP7 treatment. Furthermore, electron microscopy showed that cell shrinkage, cell wall thickening, cellular content leakage, and cell disruption were observed in the cells treated with LHP7. A gel retardation assay showed that LHP7 bound to the genomic DNA of S. aureus or plasmid DNA at a mass ratio of 2.5–10 (peptide/DNA). Circular dichroism indicated that LHP7 inserted into the groove of DNA. The cell cycle analysis showed that after the treatment with LHP7 for 30 and 60 min, the proportion of cells in I-phase increased from 8.71 to 12.09 % and from 8.71 to 15.68 %, indicating that LHP7 induced arrest of cells in the I-phase. These results would conduce to elucidate its underlying antibacterial mechanism.     

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.     

Influence on the plasma membrane of Candida albicans by HP (2-9)-magainin 2 (1-12) hybrid peptide

A 20-residue hybrid peptide (HP (2-9)-MA (1-12): HP-MA), incorporating 2-9 residues of Helicobacter pyroli ribosomal protein L1 (HP) and 1-12 residues of magainin 2 (MA), has more potent antibacterial activity than parent peptide HP (2-20) and magainin 2. In this study, the antifungal activity and its mechanism of HP-MA were investigated. HP-MA displayed a strong antifungal activity in an energy-dependent manner. To elucidate the antifungal mechanism(s) of HP-MA, FACScan analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe of Candida albicans were examined. The results indicated that the HP-MA exerts its antifungal effect by acting on the plasma membrane. Furthermore, the peptide induced remarkable morphological change when tested for membrane disrupting activity using liposomes (PC/Cholesterol; 10:1, w/w). In C. albicans, dimorphism plays a crucial role in pathogenesis but HP-MA could disrupt the mycelial forms and exert its antifungal effect on the blastoconidia in 20% fetal bovine serum.