scholarly journals Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGE

2008 ◽  
Vol 58 (2) ◽  
Author(s):  
Jasmina Ribarska ◽  
Suzana Jolevska ◽  
Ana Panovska ◽  
Aneta Dimitrovska
Keyword(s):  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Stimulating Factor

scholarly journals Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xin Xu ◽  
Koshi Asai ◽  
Daiki Kato ◽  
Kan’ichiro Ishiuchi ◽  
Kewen Ding ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Size Exclusion ◽  
High Molecular Weight Fraction ◽  
Intestinal Epithelial ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
Factor G

Abstract We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15–30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.

scholarly journals cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1

1997 ◽  
Vol 322 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Xavier F. CSAR ◽  
Alister C. WARD ◽  
Brigitte W. HOFFMANN ◽  
Graeme G. GUY ◽  
John A. HAMILTON
Keyword(s):  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Two Dimensional ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Camp Analogue ◽  
Extracellular Signal ◽  
Sds Page ◽  
Stimulating Factor

The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [γ-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF-treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.

scholarly journals Markedly Increased Vitamin B12 Concentrations Attributable to IgG–IgM–Vitamin B12 Immune Complexes

2006 ◽  
Vol 52 (11) ◽  
pp. 2107-2114 ◽  
Author(s):  
Raffick AR Bowen ◽  
Steven K Drake ◽  
Rachna Vanjani ◽  
Edward D Huey ◽  
Jordan Grafman ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Immune Complexes ◽  
Size Exclusion ◽  
Protein G ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Tof Ms

Abstract Background: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient’s serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient’s serum revealed an abnormal vitamin-B12-binding protein. SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient’s eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

Identification of Phosphoproteins Specific to Granulocyte Colony-Stimulating Factor-Mediated Signaling Using 2D-SDS-PAGE

1997 ◽  
Vol 17 (2) ◽  
pp. 77-86 ◽  
Author(s):  
XAVIER F. CSAR ◽  
ALLSTER C. WARD ◽  
BRIGITTE W. HOFFMANN ◽  
GRAEME G. GUY ◽  
JOHN A. HAMILTON
Keyword(s):  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Sds Page ◽  
Stimulating Factor

Conformational change and inactivation of arginine kinase from shrimpFeneropenaeus chinensisin oxidized dithiothreitol solutions

2004 ◽  
Vol 82 (3) ◽  
pp. 361-367 ◽  
Author(s):  
Ji-Cheng Pan ◽  
Zhen-Hang Yu ◽  
En-Fu Hui ◽  
Hai-Meng Zhou
Keyword(s):  
Conformational Change ◽  
Size Exclusion Chromatography ◽  
Tertiary Structure ◽  
Arginine Kinase ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Excitation Wavelength ◽  
Ans Binding ◽  
Sds Page ◽  
Exclusion Chromatography

The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS–PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.Key words: arginine kinase, oxidized dithiothreitol, conformational change, inactivation.

Significantly Elevated Vitamin B12 Levels in Autoimmune Lymphoproliferative Syndrome (ALPS), a Rare Lymphoproliferative Disorder with Apoptosis Defect

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4898-4898 ◽  
Author(s):  
Raffick AR Bowen ◽  
Janet K Dale ◽  
Margaret Brown ◽  
Steven K Drake ◽  
Tarsilla Moura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Serum Protein ◽  
Binding Proteins ◽  
Reference Interval ◽  
Size Exclusion ◽  
Sds Page ◽  
Exclusion Chromatography

Abstract Vitamin B12 is an essential micronutrient that plays a fundamental role in cell division and one-carbon metabolism. Vitamin B12 also plays an important role in hematopoiesis and maintaining the integrity of the nervous system. Vitamin B12 in serum is bound to two proteins, haptocorrin (HC) and transcobalamin (TC). HC is a glycosylated serum protein of ~68kDa that is produced mainly by myeloid cells and binds the majority of circulating vitamin B12 (70–90%). The exact function of this HC-bound vitamin B12 is unknown (holo-HC), but it is believed to be biologically unavailable to most cells. The remainder of vitamin B12 is bound to TC (holo-TC). TC is a 43 kDa non-glycosylated serum protein that is synthesized primarily in the enterocytes and carries vitamin B12 to the tissues via a specific receptor with a high affinity for holo-TC. Only vitamin B12 bound to TC is available for cellular uptake and use as coenzymes for L-methylmalonyl-CoA mutase and methionine synthase that synthesize succinyl-CoA and methionine, respectively. ALPS is a rare autoimmune disease in children resulting from defective lymphocyte apoptosis and is characterized by lymphoproliferation, peripheral accumulation of DNT cells (double-negative T cells; TCR alpha/beta +, CD3+ CD4− CD8−), often leading to autoimmune multilineage cytopenias. Type Ia ALPS with Fas mutations accounting for ~75% of all reported cases is the most prevalent, High serum vitamin B12 concentrations have been observed in patients with ALPS studied at NIH Clinical Center. Levels of Vitamin B12 detected in these patients range from 742 to 35365 pmol/L (median: 2489 pmol/L; mean: 4123 pmol/L; reference interval: 162–708 pmol/L) among 137 patients compiled for this study (Vitamin B12: 1pmol/L=1.355 pg/mL). To date, the mechanism of the elevated vitamin B12 concentrations in ALPS patients has not been determined and we sought to explore the source of elevated vitamin B12. Radio- and enzyme immunoassays, size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and flow cytometry techniques were used to identify and quantitate the source of the elevated vitamin B12 concentrations in peripheral blood specimens from ALPS patients and controls. Total and holo-HC levels were 36- and 32-fold higher than the upper limit of the reference interval (Total HC: 250–840 pmol/L; Holo-HC: 240–680 pmol/L), respectively, in ALPS patients. However, total and holo-TC levels (1210 pmol/L; reference interval: 500–1500 pmol/L and 143 pmol/L; reference interval: >40 pmol/L, respectively) were not elevated in ALPS patients. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins did not reveal any abnormal vitamin B12-binding proteins in ALPS. SDS-PAGE and western blot analysis revealed the presence of HC in lymphocyte lysates of ALPS but not apparently healthy controls. Flow cytometry studies also showed elevated levels of HC in all white blood cell types from ALPS patients compared to controls. The results of this study show for the first time that elevated concentrations of vitamin B12 found in ALPS patients were due to increased leukocyte (including lymphocytes) expression of HC. Further studies are needed to elucidate the mechanism for the increased synthesis and secretion of HC from lymphocytes of ALPS patients, but altered cytokine profiles, including increased interleukin-10 levels in ALPS may contribute.

scholarly journals The beta-subunit of human chorionic gonadotrophin exists as a homodimer

1999 ◽  
Vol 22 (2) ◽  
pp. 185-192 ◽  
Author(s):  
SA Butler ◽  
P Laidler ◽  
AT Kicman ◽  
T Chard ◽  
DA Cowan ◽  
...  
Keyword(s):  
Growth Factors ◽  
Size Exclusion Chromatography ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Globular Protein ◽  
Beta Subunit ◽  
Size Exclusion ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Growth Promoting

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.

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