触珠蛋白研究进展

触珠蛋白是一种酸性糖蛋白,属急性期反应蛋白之一,由于所含轻链类型的不同,触珠蛋白具有遗传多态性,触珠蛋白的合成和降解主要在肝脏进行,并受细胞因子、前列腺素、激素等的调节,触珠蛋白具有广泛的生物学功能,可能是一种重要的调节蛋白.     

Determination of the H-Y antigen in amniotic cells. Its use in prenatal diagnosis

Summary A simplified procedure is described for haptoglobin subtyping employing minute amount of serum. It consists of a partial purification of haptoglobin by cellulose-acetate electrophoresis, followed by acidic urea polyacrylamide gel electrophoresis of reduced and alkylated samples.     

Biological activities of mouse haptoglobin elicited by administration of an antitumor polysaccharide,PSK

The concentration of mouse haptoglobin in serum was increased by administration of an antitumor polysaccharide, PSK. The administration of the purified mouse haptoglobin inhibited the growth of Sarcoma-180 cells implanted in ICR mice. Furthermore, this glycoprotein enhanced macrophage activitiesin vitro, as judged from the cytostatic and cytolytic activities, glycose consumption, O₂-production, and interleukin-1 production of macrophages. In addition, mouse haptoglobin enhanced the cytolytic effect of cytotoxic T-lymphocytes. These results suggested that haptoglobin has a role in restoring or enhancing the resistance of the host against tumors.Abbreviations FCS fetal calf serum - LPS lipopolysaccharide - CTL cytotoxic T-lymphocytes - IL-1 interleukin 1 Part of this work has been presented at the 14th International Congress of Chemotherapy, Kyoto, Japan, June, 1985.     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

A general method for the complete deglycosylation of a wide variety of serum glycoproteins using peptide-N-glycosidase-F

Many indirect serum studies show changes in protein glycosylation in disease, but further progress will require direct investigation of oligosaccharide composition. Current methods of deglycosylation using PNGase-F often result in incomplete removal of oligosaccharides. This is an unsatisfactory situation because only small quantities of material are often available in clinical studies, glycosylation changes may occur in only a small proportion of the molecules and quantification of the released oligosaccharides may be unreliable. The ability of PNGase-F to deglycosylate haptoglobin (Hp) under different conditions has been investigated. Oligosaccharides were completely removed from 50g of Hp by treatment for 24 h with PNGase-F in 50 mmol/l ammonium formate buffer, pH 8.6, in combination with sodium dodecyl sulphate, mercaptoethanol and Nonidet P40. This modified procedure was equally effective at removing oligosaccharides from other serum glycoproteins (₁ glycoprotein, ₁, transferrin) and fetuin, and its efficiency was independent of the polymeric structure of the molecule or the amount of glycosylation. The method has the additional advantage of using 20% less enzyme than previous methods, which substantially reduces costs.     

Subtyping of haptoglobin — Presentation of a new method

Summary A method is described for large scale routine phenotyping of haptoglobin (Hp) which allows complete subtyping without prior purification of the Hp molecule. The procedure includes polyacrylamide gel isoelectric focusing of reduced, neuraminidase treated serum or plasma samples, and nitrocellulose blots developed with the immunoperoxidase technique. Different variables including sample treatment, electrofocusing, blotting procedures, and immunoperoxidase visualization are discussed.Characteristic -chain patterns allow identification of the common allotypes 2FS, 2SS, 2FF, IS, IF, and Johnson. Isoelectric variations in the -chain may also be recognized. For comparison, two-dimensional Hp-patterns are presented. The results from concurrent typing of 600 samples by ordinary starch gel electrophoresis and by the described isofocusing technique, are evaluated.     

Multiple immunoreactive forms of calcitonin in human plasma

Purified rabbit serum haptoglobin was partially characterized, and it was found that the hemoglobin-binding property and subunit structure were similar to those of human type 1-1, swine, canine, and equine haptoglobins. However, rabbit haptoglobin was dissociated into subunits and few intermediates only in the presence of urea or sodium dodecyl sulfate without reduction. Thus the absence of interchain disulfide bonds in rabbit haptoglobin is unique among many animal haptoglobins.     

Utilization of myoglobin as a heme source by Haemophilus influenzae requires binding of myoglobin to haptoglobin

Haemophilus influenzae has an absolute growth requirement for heme. One potential in vivo source of heme is the protein myoglobin which is found at low levels in human serum. No tested H. influenzae strain was able to use myoglobin as a heme source. However, all strains were able to utilize the heme from myoglobin when myoglobin was complexed with haptoglobin. Utilization of the haptoglobin-myoglobin complex was shown to be mediated by the previously described hemoglobin/hemoglobin-haptoglobin-binding proteins of H. influenzae.     

Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames

AIMS: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. METHODS AND RESULTS: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). CONCLUSIONS: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. SIGNIFICANCE AND IMPACT OF THE STUDY: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.     

Differential utilization by Haemophilus influenzae of haemoglobin complexed to the three human haptoglobin phenotypes

Haemophilus influenzae has an absolute requirement for heme, which may be supplied as the haemoglobin-haptoglobin complex. Utilization of haemoglobin-haptoglobin by H. influenzae is mediated by a family of proteins termed the haemoglobin-haptoglobin binding proteins (Hgps), of which a given strain may contain up to four genes. Human haptoglobin occurs in three phenotypes (1-1, 2-1 and 2-2). Using mutant derivatives of an H. influenzae type b strain that expressed single Hgps we analysed the ability of each Hgp to utilize haemoglobin complexed to the various haptoglobin phenotypes. A strain expressing only HgpB was able to utilize haemoglobin bound to all haptoglobin phenotypes significantly better than strains expressing either HgpA or HgpC.     

Glycosylation and over-expression of endometriosis-associated peritoneal haptoglobin

Peritoneal endometriotic tissues synthesize and secrete haptoglobin (pHp), which has an analogous nucleotide sequence to hepatic haptoglobin found in serum (sHp). This study performed enzymatic digestions and lectin binding assays to determine if differences in protein glycosylation exist between sHp and pHp, which may provide insight into pHp function and/or identify epitopes for development of novel methods of medical management of endometriosis. To reduce the dependence on surgical collection of peritoneal tissues from women, recombinant peritoneal Hp (rpHp) was produced and its glycosylation analyzed for future functional studies. These results showed the apparent molecular weight of pHp was 3 kDa smaller than sHp. Desialylation and complete N-deglycosylation elicited similar shifts in sHp and pHp electrophoretic migration, suggesting similar sialic acid content and indicating the 3 kDa variance was due to carbohydrate content, not protein degradation, respectively. Sequential deglycosylation of the four sHp N-glycan chains caused a 3 kDa shift per N-glycan removed suggesting the 3 kDa difference between sHp and pHp may be one N-glycan chain. Lectin ELISA and lectin-blotting analyses demonstrated increased pHp and rpHp interactions with MAL and LTL but no difference in binding to SNL compared to sHp from healthy individuals, identifying variations in the ratios of (2-3) to (2-6) sialic acid and fucose residues. Recombinant pHp was 100-fold over-expressed with a similar glycosylation pattern to pHp, albeit in an unprocessed - Hp polypeptide form. These results are the first to identify differences between pHp and sHp glycosylation and lay groundwork further studies to characterize anomalies in glycan composition and structure, which likely impart pHp with known immunomodulatory functions and may be used as epitopes for development of immune based therapeutics for novel, non-surgical management of endometriosis.     

Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient''s plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient''s will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.     

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.     

Elevated levels of circulating pancreatic polypeptide in inflammatory and infectious disorders

Circulating levels of pancreatic polypeptide (PP) were found to be elevated when compared to healthy controls in 54% of patients with chronic inflammatory connective tissue disorders (SLE, rheumatoid arthritis, scleroderma, mixed connective tissue disease and temporal arteritis) and in 96% of patients with acute viral or bacterial infections. Significant positive correlations were obtained between the serum values of PP and those of haptoglobin or orosomucoid. Accompanying successful anti-inflammatory treatment of patients with autoimmune disorders, a reduction of PP levels was observed. The findings suggest that the magnitude of increase in PP was associated with the degree of the inflammatory activity. Raised PP levels may contribute to the alterations in carbohydrate and lipid metabolism observed during active inflammatory diseases in man.     

A population genetic survey of the haptoglobin polymorphism in Melanesians by DNA analysis.

We have determined the haptoglobin (Hp) genotypes of 831 Melanesians from Vanuatu, Papua New Guinea, and New Caledonia by Southern blot analysis of DNA extracted from umbilical cord and peripheral blood samples. There was complete agreement between these genotypes and the protein phenotype in cases where both were determined, and genotyping was possible in cases where no serum haptoglobins were measurable. Subtyping of Hp1 alleles using restriction enzymes showed that Melanesians, like Mongoloids and Australian Aboriginals, have only the Hp1S allele. Three cases of Hp Johnson were found in Vanuatu, and further restriction mapping supported a partial gene triplication model for the structure of this variant. We also report a new common BclI restriction enzyme polymorphism upstream of the Hp1 gene. The advantages of using DNA for haptoglobin typing are discussed.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.     

Mass spectrometric assay for analysis of haptoglobin fucosylation in pancreatic cancer

A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10 μL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated triantennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer.     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

Simple method for the preparation of haptoglobin]

The present paper deals with a method permitting the isolation of haptoglobin 2-2 from human serum or plasma. The haptoglobin is adsorbed to DEAE-cellulose at pH 5.1 by batching. The loaded cellulose is given into a column and the haptoglobin eluted by a 0.1 M to 0.15 M acetate buffer. The last step is a gel-chromatography on Sephadex G-200. Disc- and immunoelectrophoresis were used to test the purity. As by-product acid alpha1-glycoprotein can be obtained.     

Status and metabolism of iron in elite sportsmen during a period of professional competition

The aim of this study was to determine the effect of both acute exercise and maintained training during a period of competition (3 mo, at the start of the season) on iron metabolism in sportsmen on a professional volleyball team. Twelve sportsmen volunteered for this study. The exercise test was performed on a mechanically braked Monark cycle ergometer and consisted of a triangular progressive test. Three blood samples were obtained in each test: at rest, just after exercise, and after recovery. The following hematological parameters were determined: red blood count (RBC), hemoglobin (Hb) and hematocrit (Hto), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total proteins (TP), serum iron (Fe) and total iron-binding capacity (TIBC), ferritin (FER), transferrin (TRF), haptoglobin (HPT), and serum cortisol (COR) concentrations. We have found changes in hematological and biochemical variables related to Fe metabolism during the study. The changes observed could be the result of hemoconcentration processes after exercise and, at least in part, to physical stress and muscular damage. We conclude that athletes, after a period of adaptation, with a good plan of work/recovery series, undergo a biological redistribution on hematological and biochemical parameters concerning Fe metabolism during the training and competition period. Also, daily Fe supplementation could restore and mask the true repercussions of maintained training observed in other sports.