Serum amyloid A, an acute phase protein, inhibits platelet activation.

The effect of serum amyloid A, an acute phase protein, on platelet function was studied. Serum amyloid A was isolated and purified from sera of patients with recent trauma. Serum amyloid A inhibited thrombin-induced gel-filtered platelet aggregation. However, it did not inhibit aggregation induced by collagen or adenosine diphosphate, nor did it influence the aggregation of platelet-rich plasma activated with thrombin. Further studies of its effect on thrombin-induced activities showed that serum amyloid A, at concentrations of 25 to 100 micrograms/ml (which are found in mild acute events), suppressed the increase of cytosolic [Ca2+], thromboxane generation, and carbon 14-labeled serotonin release in a dose-dependent fashion. Serum amyloid A did not interfere with the clotting or amidolytic activities of thrombin. Therefore, the data suggest a protective role for serum amyloid A in thromboembolic disease by specific interaction with thrombin-induced platelet activation. Amyloid A protein also markedly inhibited thrombin-induced platelet aggregation. Because amyloid A is homologous to the N-terminal portion of serum amyloid A, the observed activity probably resides in that part of the molecule. This finding may be of importance in localization of the active site on serum amyloid A.     

Transthyretin inhibition of amyloid beta aggregation and toxicity

OBJECTIVES: The aim of this study was to investigate transthyretin (prealbumin) effects on Abeta25-35-induced cytotoxicity. DESIGN AND METHODS: In view of the well-recognized literature data demonstrating that Abeta25-35 fibrillar aggregates cause in vitro cytotoxicity to human red blood cells and apoptotic changes to SK-N-BE neuroblastoma cells in cultures (ultrastructural evidence), we tested transthyretin effects on these two experimental models. RESULTS: Incubation of Abeta25-35 with transthyretin (at transthyretin concentrations equal to CSF physiological levels) demonstrated both inhibition of red blood cells lysis and neutralization of SK-N-BE neuroblastoma cells ultrastructural apoptotic changes. Moreover, transthyretin was shown to be able to inhibit the formation of fibrillar macroaggregates of Abeta25-35. CONCLUSIONS: The findings imply that experimental systems investigating Abeta-induced cytotoxicity consider the protective interaction of transthyretin with Abeta; an interaction to be considered also in vivo in view of the fact that transthyretin immunoreactivity has been previously demonstrated in amyloid plaques of brains from Alzheimer's disease patients.     

西红柿汁体外抑制血小板聚集功能及其机制探讨

目的:观察西红柿汁在体外对血小板聚集、血小板活化标志物的表达及对血小板纤维蛋白原结合量的影响,初步探讨西红柿汁对血小板聚集功能的影响机制。方法:将不同浓度的西红柿汁与正常人富血小板血浆(PRP)孵育,加入5-腺苷二磷酸二钠盐(ADP)和胶原,观察西红柿汁对血小板聚集的影响。用ADP及胶原激活血小板,流式细胞仪(FCM)分析西红柿汁对活化血小板纤维蛋白原受体(Fib-R)和P-选择素(CD62P)的表达水平及血小板纤维蛋白原结合量的影响。结果:西红柿汁能够明显抑制由ADP、胶原诱导的血小板聚集,且随着西红柿汁浓度的增加,对血小板聚集的抑制率增高。西红柿汁不能抑制ADP、胶原诱导的血小板Fib-R、CD62P表达,但能显著抑制由ADP、胶原诱导的血小板与纤维蛋白原结合量。结论:西红柿汁在体外能够显著抑制血小板聚集,其抗血小板聚集的作用机制与阻碍纤维蛋白原与血小板Fib-R结合有关。西红柿汁具有潜在的抗血小板治疗前景。     

Effect of resveratrol, a polyphenolic phytoalexin, on thermal hyperalgesia in a mouse model of diabetic neuropathic pain

Diabetic neuropathic pain, an important microvascular complication in diabetes mellitus, has been recognized as one of the most difficult types of pain to treat. The underlying mechanisms of painful symptoms may be closely associated with hyperglycaemia but a lack of the understanding of its proper aetiology, inadequate relief, development of tolerance and potential toxicity of classical antinociceptives warrant the investigation of the newer agents to relieve this pain. The aim of the present study was to explore the antinociceptive effect of resveratrol on diabetic neuropathic pain and to examine its effect on serum tumour necrosis factor-alpha (TNF-alpha) and whole brain nitric oxide (NO) release. Four weeks after a single intraperitoneal injection of streptozotocin (STZ, 200 mg/kg), mice were tested in the tail immersion and hot-plate assays. Diabetic mice exhibited significant hyperalgesia along with increased plasma glucose and decreased body weights when compared with control mice. Daily treatment with resveratrol (5, 10 and 20 mg/kg body weight; p.o.) for 4 weeks starting from the 4th week of STZ injection significantly attenuated thermal hyperalgesia. Resveratrol also decreased the serum TNF-alpha levels and whole brain NO release in a dose-dependent manner. These results point towards the potential of resveratrol in attenuating diabetic neuropathic pain.     

Elusive function of prion protein

Prion protein is a highly conserved glycoprotein tethered to cell membranes by a glycosylphosphatidylinositol(GPI) anchor that is expressed in many tissues including brain, heart, and muscle. Although misfolding of the cellular prion protein (PrP(c)) into alternative form, denoted (PrP(Sc)), is a key event in prion infections, the normal function of PrPc remains to be clearly defined. Many PrP(c)-binding proteins have been identified, and several roles for PrP(c) have been suggested, including oxidative stress, cell adhesion, copper uptake, cell survival, protection against oxidative stress, but authentication of these interactions in functional assays is incomplete. In this article, we pick out some researches that pertain to the biology of mammalian prion protein functions.     

A sensitive immunochemiluminescence assay for human serum amyloid A protein

We describe an immunochemiluminescence assay for human plasma serum amyloid A protein (SAA) in which specific rabbit polyclonal antibodies against synthetic peptides are used. The detection of the antigen-antibody reaction at 425 nm is based on a brief emission of light by a luminophor component (signal) in response to chemical energy. The working range of the assay covers plasma SAA concentrations from 5 to 100 micrograms/L. The lower detection limit is 5 micrograms/L, the within- and between-assay CVs are less than 12%. Bilirubin, cholesterol and triglyceride in final concentrations of up to 220 mumol/L, 8.1 mmol/L and 2.68 mmol/L, respectively, do not interfere with the assay. Results were correlated with those obtained by the enzyme-linked immunosorbent assay using the same antibodies (r = 0.95; p less than 0.001; n = 50). This method is inexpensive, simple and easily automated.     

His18 promotes reactive oxidative stress production in copper-ion mediated human islet amyloid polypeptide aggregation

Copper ions play a critical role in human islet amyloid polypeptide (hIAPP) aggregation, which has been found in more than 90% of patients with type-2 diabetes (T2D). The role of Cu(ii) in the cell cytotoxicity with hIAPP has been explored in two aspects: inhibiting the formation of fibrillar structures and stimulating the generation of reactive oxygen species (ROS). In this work, we carried out spectroscopic studies of Cu(ii) interacting with several hIAPP fragments and their variants as well. Electron paramagnetic resonance (EPR) measurements and Amplex Red analysis showed that the amount of H₂O₂ generated in hIAPP(11-28) solution co-incubated with Cu(ii) was remarkably more than hIAPP(1-11) and hIAPP(28-37). Furthermore, the H₂O₂ level was seriously reduced when His18 of hIAPP(11-28) was replaced by Arg(R) or Ser(S), indicating that His18 is the key residue of Cu(ii) binding to hIAPP(11-28) to promote H₂O₂ generation. This is likely because the donation of electrons from the peptide to Cu(ii) ions would result in the formation of the redox-active complexes, which could stimulate the formation of H₂O₂. Overall, this study provides further insight into the molecular mechanism of Cu(ii) induced ROS generation.

His18 promotes H₂O₂ production in copper-ion mediated hIAPP aggregation.     

Underestimation of the expression of cellular prion protein on human red blood cells

BACKGROUND: Recent transmissions of variant Creutzfeldt‐Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP^C) in both plasma and blood cells. According to published studies, most of PrP^C in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP^C associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme‐linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP^C per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP^C. Binding of alternate PrP^C MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP^C per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP^C was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP^C conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP^C/cell, which makes them, due to high RBC numbers, major contributors to the pool of cell‐associated PrP^C in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP^C in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP^C.     

重组人干细胞因子/血小板生成素融合蛋白的纯化及复性研究

目的　探讨重组人干细胞因子/血小板生成素(SCF TPO)融合蛋白纯化及复性的最佳方法。方法　裂解 法提取包涵体,在变性条件下利用金属螯和层析获得融合蛋白,经透析及谷胱甘肽氧化还原法对纯化的融合蛋白进 行复性。结果　获得具有初步生物学活性的SCF TPO融合蛋白,其纯度高达90%。结论　该纯化方法操作简捷, 特异性高,纯化效果好,获得率高。     

Regulation of divalent metal ions to the aggregation and membrane damage of human islet amyloid polypeptide oligomers

The accumulation of human islet amyloid polypeptide (hIAPP) on the surface of pancreatic β cells is closely related to the death of the cells. Divalent metal ions play a significant role in the cytotoxicity of hIAPP. In this study, we examined the roles played by the divalent metal ions of zinc, copper and calcium in the aggregation of both hIAPP18-27 fragment and full-length hIAPP and the ability of their oligomers to damage the membrane of POPC/POPG 4 : 1 LUVs using the ThT fluorescence, TEM, AFM, CD, ANS binding fluorescence and dye leakage experiments. We prepared metal-free and metal-associated oligomers that are similar in size and aggregate slowly using the short peptide and confirmed that the ability of the peptide oligomers to damage the lipid membrane is reduced by the binding to the metal ions, which is closely linked to the reducing hydrophobic exposure of the metal-associated oligomers. The study on the full-length hIAPP showed that the observed membrane damage induced by hIAPP oligomers is either mitigated at a peptide-to-metal ratio of 1 : 0.33 or aggravated at a peptide-to-metal ratio of 1 : 1 in the presence of Zn(ii) and Cu(ii), while the surface hydrophobicity of hIAPP oligomers was reduced at both peptide-to-metal ratios. The observed results of the membrane damage were attributed to the counteraction between a decrease in the disruptive ability of metal-associated oligomer species and an increase in the quantity of oligomers promoted by the binding of the metal ions to hIAPP oligomers. The former could play a predominant role in reducing the membrane damage at a peptide-to-metal ratio of 1 : 0.33, while the latter could play a predominant role in enhancing the membrane damage at a peptide-to-metal ratio of 1 : 1. This study shows that an enhanced membrane damage could be caused by the oligomer species with a decreased instead of an increased disruptive ability, given that the abundance of the oligomer species is high enough.

Their is a counteraction between a decrease in the disruptive ability of metal-associated oligomer species and an increase in the quantity of oligomers promoted by the metal binding in the activity of hIAPP induced membrane damage.     

Divergent expression of cellular prion protein on blood cells of human and nonhuman primates

BACKGROUND: Four recent transmissions of variant Creutzfeldt-Jakob disease infection by transfusion highlight the need for detailed understanding of blood-related prion pathogenesis. Nonhuman primates are the most relevant models of human prion diseases. STUDY DESIGN AND METHODS: Quantitative flow cytometry with monoclonal antibodies FH11, 3F4, and 6H4 against different parts of the normal cellular form of the prion protein (PrP(C)) was used to evaluate its expression on blood cells of humans, chimpanzees, cynomolgus macaques, rhesus macaques, squirrel monkeys, and microcebe lemurs. RESULTS: Chimpanzees, rhesus macaques, and squirrel monkeys displayed a much higher quantity of total blood cell membrane PrP(C) than humans, due to a markedly higher expression of PrP(C) on their red blood cells (RBCs). In contrast, cynomolgus macaques and lemurs demonstrated substantially lower levels of membrane PrP(C) due to the lack of significant PrP(C) expression on RBCs and platelets (PLTs). All species displayed PrP(C) on white blood cells (WBCs), with the highest levels found on human cells. Only humans, chimpanzees, and to a lesser degree rhesus macaques expressed PrP(C) on PLTs. CONCLUSION: If PrP(C) contributes to the propagation or transport of prion infectivity in blood, the differences reported here need to be considered when extrapolating results of transmission studies in primate models to blood and blood components in humans.     

Characterization of an amyloid fibril protein from senile cardiac amyloid

A protein, ASCA, is isolated from amyloid fibrils extracted from heart tissue of five different patients with senile cardiac amyloidosis (SCA). The proteins of all five patients showed immunological identity when reacted with an antiserum raised against one of the proteins. In contrast, no reaction was obtained with antisera against a variety of other amyloid proteins. The antiserum against the subunit protein of senile cardiac amyloid did not react with any other amyloid preparations tested, nor with extracts of normal heart tissue. Thus, the subunit protein appeared to be unique to senile heart amyloid. The protein could form fibrils in vitro, had a mol wt of about 6,000 daltons and the amino acid compositions investigated in two cases showed extensive similarities but were clearly different from that of protein AA of secondary amyloid fibrils.     

Transfusion transmission of human prion diseases

No transmission through transfusion has been reported for classic Creutzfeldt-Jakob disease (CJD). Moreover, a series of epidemiological surveillance, case-control, and look-back studies have provided no evidence of such transmission of CJD. Hence, the risk of such transfusion transmission of classic CJD remains theoretical. In contrast, based on data from the United Kingdom, the likelihood of transmission of the agent of the variant form of CJD (vCJD) through blood transfusion by donors who develop the disease within several years of donation is about 14% for recipients who survive longer than 5 years posttransfusion. Leukodepletion may reduce the likelihood of vCJD transmissions, although this procedure by itself removes less than half of the prion infectivity of blood. The potentially longer incubation periods of vCJD with infections in donors who are not methionine/methionine homozygous at codon 129 of the prion protein gene, the unknown number of such donors, and the unknown infectivity of their blood during the incubation period suggests caution in assuming that only known cases of vCJD represent a risk for the transfusion transmission of vCJD. Results from ongoing look-back investigations and other studies will enable continued monitoring and more precise estimations of the risks of the transfusion transmission of CJD and vCJD.     

Degradation of serum amyloid A protein by surface-associated enzymes of human blood monocytes

Peripheral blood monocytes incubated in a serum-free medium degraded serum amyloid A (SAA) protein along three pathways. Of 20 normal subjects, 8 degraded SAA completely with no detectable intermediates. Eight subjects transiently produced an amyloid A (AA)-like intermediate which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) with tissue AA protein and reacted with antisera to AA, whereas four subjects yielded a persistent AA-like intermediate on PAGE. This group also failed to degrade tissue AA protein. Cells from 10 patients with amyloidosis fell into the second group. The responsible enzymes appear to be serine proteases because they are inhibited by disopropyl fluorophosphate. They were not affected by epsilon-amino caproic acid, L-1-tosylamide-2-phenylethyl chloromethyl ketone, or N-alpha-p-tosyl-L-lysine chlormethyl ketone. It appears possible that the enzymes are associated with the outer membrane of the cell because only a small fraction of the activity is secreted into the medium and because enzyme activity remains after fixation of the cells with glutaraldehyde which completely stops phagocytosis. Perhaps differences in patterns of proteolysis may play a role in the predisposition to amyloidosis.     

Comparative analysis of normal prion protein expression on human,rodent, and ruminant blood cells by using a panel of prion antibodies

BACKGROUND: It is not known whether variant CJD can be transmitted within the human population by blood transfusion. The expression of normal cellular prion protein (PrPC) by different blood cell types may permit selective uptake and dissemination of infectivity. STUDY DESIGN AND METHODS: The normal distribution of PrPC on the major blood cell types of species known to be susceptible to natural or experimental transmissible spongiform encephalopathies was studied. Blood from healthy humans, mice, hamsters, cattle, and sheep was examined by flow cytometry by using a large panel of antibodies with different prion protein (PrP) epitope specificities to maximize the detection of PrP variants across species and cell type. RESULTS: PrP was detected on all major human blood cells types except eosinophils, but was not detected as ubiquitously or uniformly on major blood cell types of different animal species. CONCLUSION: Different animal species have unique patterns of expression of PrPC on blood cell types, with none equivalent to the human pattern. This needs to be considered when extrapolating from animal models of blood-borne transmissible spongiform encephalopathy infectivity, particularly in regard to the risk assessment of potential variant CJD spread within the human population. The relationship between PrP distribution and infectivity distribution in blood needs further investigation.     

The carbohydrate composition of human serum amyloid P component

The carbohydrate moiety of human serum amyloid P component was analyzed and found to consist of equal amounts of galactose and mannose (total 4.0%), of glucosamine and galactosamine in a ratio of 7:1 (total 2.7%) and sialic acid (3.9%). It should be noted that this is the first report on the separate quantification of the neutral hexoses and the demonstration of the presence of galactosamine. The contents of glucosamine and galactosamine suggest that this protein possesses both an N- and an O-glycan.     

重组人生长激素对胸部外伤老年患者蛋白质代谢的作用

目的研究应用重组人生长激素(rhGH)对接受常规肠内营养支持的胸部外伤老年患者蛋白质代谢的调理作用。方法选择20例多发性肋骨骨折(肋骨骨折3根以上)、年龄大于70岁的老年患者20例,随机分为两组。治疗组10例,采用标准肠内营养+rhGH(rhGH每天12U皮下注射,共计8d);另外10例作为对照组,仅应用肠内营养治疗。两组患者其他治疗完全相同。治疗前和治疗后的第8天,分别测定血清白蛋白、血清转铁蛋白、血清前白蛋白浓度和双手握力,统计医院获得性肺炎的发生情况。结果治疗组患者在治疗后第8天的血清白蛋白、血清转铁蛋白和血清前白蛋白水平比对照组显著升高(P<0.05),双手握力与对照组相比有显著增强(P<0.01);治疗组患者医院获得性肺炎的发生率也较对照组显著降低(P<0.05)。结论老年胸部外伤患者蛋白质的合成能力较差,在肠内营养支持下加用rhGH可以明显促进老年患者蛋白质合成代谢,增加肌肉的收缩力,增强排痰力度,减少医院获得性肺炎的发生率。