Affinity to lectin, biological and immunological characteristics of human chorionic gonadotropins from pregnant women and trophoblastic tumour patients

1986 ◽  
Vol 112 (4) ◽  
pp. 579-585 ◽  
Author(s):  
Koji Koyama ◽  
Kazushi Toda ◽  
Daisaku Kuriyama ◽  
Shinzo Isojima
Keyword(s):  
Affinity Chromatography ◽  
Pregnant Women ◽  
Hydatidiform Mole ◽  
Affinity Column ◽  
Unbound Fraction ◽  
Lentil Lectin ◽  
In Vitro Bioassays ◽  
Trophoblastic Tumours

Abstract. To compare the affinity to lectin of human chorionic gonadotropins (hCG) from pregnant women and trophoblastic tumour patients, a small amount of urine was fractionated by a lentil lectin (LcH) affinity chromatography. The LcH-bound fractions were eluted with 2% mannoside solution, and each fraction was assayed for hCG activities by radioimmunoassay. In pregnant women, more than 90% of hCG immunoactivity in urine was bound to the LcH column and eluted from it, whereas 8 to 26% and 37 to 51% of the activity were not adsorbed to the affinity column and were recovered in the LcH-unbound fraction in the patients with hydatidiform mole and choriocarcinoma, respectively. These results suggest that LcH affinity chromatography of urinary hCG contributes to differential diagnosis between pregnancy and trophoblastic tumours. To characterize the properties of hCG from the urine of choriocarcinoma patients, with or without the LcH affinity, hCG activities in both LcH-unbound and LcH-bound fractions were measured by in vivo and in vitro bioassays and each of the activities was compared with the activities measured by radioimmunoassay. The results showed that the LcH-unbound fraction contained hCG molecules defecting to induce the biological activity of hCG in vivo.

scholarly journals Reductions of copper ion-mediated low-density lipoprotein (LDL) oxidations of trypsin inhibitors, the sweet potato root major proteins, and LDL binding capacities

2020 ◽  
Vol 61 (1) ◽  
Author(s):  
Yeh-Lin Lu ◽  
Chia-Jung Lee ◽  
Shyr-Yi Lin ◽  
Wen-Chi Hou
Keyword(s):  
Sweet Potato ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Trypsin Inhibitors ◽  
Water Soluble ◽  
Affinity Column ◽  
Low Density ◽  
Native Page

Abstract Background The root major proteins of sweet potato trypsin inhibitors (SPTIs) or named sporamin, estimated for 60 to 80% water-soluble proteins, exhibited many biological activities. The human low-density lipoprotein (LDL) showed to form in vivo complex with endogenous oxidized alpha-1-antitrypsin. Little is known concerning the interactions between SPTIs and LDL in vitro. Results The thiobarbituric-acid-reactive-substance (TBARS) assays were used to monitor 0.1 mM Cu2+-mediated low-density lipoprotein (LDL) oxidations during 24-h reactions with or without SPTIs additions. The protein stains in native PAGE gels were used to identify the bindings between native or reduced forms of SPTIs or soybean TIs and LDL, or oxidized LDL (oxLDL). It was found that the SPTIs additions showed to reduce LDL oxidations in the first 6-h and then gradually decreased the capacities of anti-LDL oxidations. The protein stains in native PAGE gels showed more intense LDL bands in the presence of SPTIs, and 0.5-h and 1-h reached the highest one. The SPTIs also bound to the oxLDL, and low pH condition (pH 2.0) might break the interactions revealed by HPLC. The LDL or oxLDL adsorbed onto self-prepared SPTIs-affinity column and some components were eluted by 0.2 M KCl (pH 2.0). The native or reduced SPTIs or soybean TIs showed different binding capacities toward LDL and oxLDL in vitro. Conclusion The SPTIs might be useful in developing functional foods as antioxidant and nutrient supplements, and the physiological roles of SPTIs-LDL and SPTIs-oxLDL complex in vivo will investigate further using animal models.

Expression, purification and properties of multidrug efflux proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 513-517 ◽  
Author(s):  
P.J. F. Henderson ◽  
C. K. Hoyle ◽  
A. Ward
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
General Strategy ◽  
X Rays ◽  
Multidrug Efflux ◽  
Nmr Studies ◽  
E Coli ◽  
Efflux Proteins

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H+ antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1–25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni2+-nitrilo-triacetate-affinity column chromography, yielding 5–25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of α-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with 13C or 15N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

scholarly journals Proposal of a Pharmacokinetically Optimized Dosage Regimen of Antibiotics in Patients Receiving Continuous Hemodiafiltration

2011 ◽  
Vol 55 (12) ◽  
pp. 5804-5812 ◽  
Author(s):  
Takehito Yamamoto ◽  
Nobuhiro Yasuno ◽  
Shoichi Katada ◽  
Akihiro Hisaka ◽  
Norio Hanafusa ◽  
...  
Keyword(s):  
Critically Ill ◽  
Critically Ill Patients ◽  
Drug Clearance ◽  
Minor Effect ◽  
Optimal Dosage ◽  
Continuous Hemodiafiltration ◽  
Unbound Fraction ◽  
Artificial Plasma

ABSTRACTThe aim of the study was to quantitatively predict the clearance of three antibiotics, amikacin, vancomycin, and teicoplanin, during continuous hemodiafiltration (CHDF) and to propose their optimal dosage in patients receiving CHDF. For this goal,in vitroCHDF experiments with a polyacrylonitrile (PAN) membrane were first performed using these antibiotics, and then the clearances were compared within vivoCHDF situations determined in 16 critically ill patients. Thein vitroCHDF clearances were described as the product of the outflow rate of a drain (Qoutflow) and the drug unbound fraction in artificial plasma, indicating that drug adsorption to the PAN membrane has minor effect on drug clearance in our settings. The observedin vivoclearances also agreed very well with the predicted values, with a product ofQoutflowand plasma unbound fraction, when residual creatinine clearance (CLCR) was taken into account (within a range of 0.67- to 1.5-fold for 15 of 16 patients). Based on these results, a nomogram of the optimized dosages of amikacin, vancomycin, and teicoplanin was proposed, and it was evident thatQoutflowand residual CLCRare major determinants of the dosage and dosing interval for these antibiotics. Although the applicability needs to be confirmed with another type of membrane or higherQoutflow, our nomogram can help determine the dosage setting in critically ill patients receiving CHDF.

scholarly journals Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1784-1789 ◽  
Author(s):  
E Niskanen ◽  
J Gorman ◽  
PC Isakson
Keyword(s):  
B Cells ◽  
Colony Formation ◽  
High Performance ◽  
B Lymphocyte ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Plasma Clot ◽  
Lentil Lectin

Abstract In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3271-3271
Author(s):  
Teresa M Brophy ◽  
Jasimuddin Ahamed ◽  
Barry S. Coller
Keyword(s):  
Transforming Growth Factor ◽  
Coagulation Factor ◽  
Transforming Growth Factor Β1 ◽  
Affinity Column ◽  
Factor V ◽  
Disulfide Exchange ◽  
Control Peptide ◽  
Tgf Β1

Abstract Abstract 3271 Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation. Disclosures: Coller: Centocor/Accumetrics/Rockefeller University:.

scholarly journals Comparative Efficacies of Human Simulated Exposures of Telavancin and Vancomycin against Methicillin-Resistant Staphylococcus aureus with a Range of Vancomycin MICs in a Murine Pneumonia Model

2010 ◽  
Vol 54 (12) ◽  
pp. 5115-5119 ◽  
Author(s):  
Jared L. Crandon ◽  
Joseph L. Kuti ◽  
David P. Nicolau
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Load ◽  
Infection Model ◽  
Time Curve ◽  
Unbound Fraction ◽  
Methicillin Resistant ◽  
Vancomycin Mics ◽  
Mrsa Strains

ABSTRACT Telavancin displays potent in vitro and in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA), including strains with reduced susceptibility to vancomycin. We compared the efficacies of telavancin and vancomycin against MRSA strains with vancomycin MICs of ≥1 μg/ml in a neutropenic murine lung infection model. Thirteen clinical MRSA isolates (7 vancomycin-susceptible, 2 vancomycin-heteroresistant [hVISA], and 4 vancomycin-intermediate [VISA] isolates) were tested after 24 h, and 7 isolates (1 hVISA and 4 VISA isolates) were tested after 48 h of exposure. Mice were administered subcutaneous doses of telavancin at 40 mg/kg of body weight every 12 h (q12h) or of vancomycin at 110 mg/kg q12h; doses were designed to simulate the area under the concentration-time curve for the free, unbound fraction of drug (fAUC) observed for humans given telavancin at 10 mg/kg q24h or vancomycin at 1 g q12h. Efficacy was expressed as the 24- or 48-h change in lung bacterial density from pretreatment counts. At dose initiation, the mean bacterial load was 6.16 ± 0.26 log10 CFU/ml, which increased by averages of 1.26 ± 0.55 and 1.74 ± 0.68 log in untreated mice after 24 and 48 h, respectively. At both time points, similar CFU reductions were noted for telavancin and vancomycin against MRSA, with vancomycin MICs of ≤2 μg/ml. Both drugs were similarly efficacious after 24 and 48 h of treatment against the hVISA strains tested. Against VISA isolates, telavancin reduced bacterial burdens significantly more than vancomycin for 1 of 4 isolates after 24 h and for 3 of 4 isolates after 48 h. These data support the potential utility of telavancin for the treatment of MRSA pneumonia caused by pathogens with reduced susceptibility to vancomycin.

Synthetic antagonists of the myometrial response to vasopressin and oxytocin

1986 ◽  
Vol 111 (1) ◽  
pp. 125-131 ◽  
Author(s):  
P. Melin ◽  
J. Trojnar ◽  
B. Johansson ◽  
H. Vilhardt ◽  
M. Åkerlund
Keyword(s):  
Blood Pressure ◽  
Pregnant Women ◽  
Solid Phase ◽  
Phase Method ◽  
Inhibitory Effects ◽  
Inhibitory Potency ◽  
Low Blood Pressure ◽  
Solid Phase Method

ABSTRACT With the aim of developing inhibitors of vasopressin-and oxytocin-induced uterine activity, 17 analogues of 1-deamino-oxytocin were synthesized by the solid-phase method. Modifications were made at positions 2, O-methyltyrosine (Tyr(OMe)) and O-ethyltyrosine (Tyr(OEt)),d-Tyr,d-Tyr(OEt),d-Trp; 4, Val,Thr and 8, Orn,Cit,Arg,d-Arg. The analogues were tested for antiuterotonic activity in vitro and in vivo in the rat and in vitro on myometrial strips from non-pregnant women and pregnant women at term. Their selectivity was also investigated in blood pressure and antidiuretic bioassays in rats. Results were compared with those from an original antiuterotonic analogue 1-deamino-2-Tyr(OEt)-oxytocin (d(OEt)-oxytocin). In the rat in vitro and in vivo all analogues possessed higher antiuterotonic activity than d(OEt)-oxytocin. The negative logarithm of the molar concentration of the antagonist which reduced the effect of a dose of agonist to that of half the dose (pA2) was between 7·6 and 8·9 for all the new inhibitors compared with 7·2 for d(OEt)-oxytocin. The highest pA2 value was found for 1-deamino-2-Tyr(OMe)-8-Orn-oxytocin (8·9 ± 0·2, s.e.m.) and 1-deamino-2-Tyr(OEt)-4-Thr-8-Orn-oxytocin (8·9 ± 0·6). In myometrium from non-pregnant women the most potent peptide was 1-deamino-2-d-Tyr(OEt)-4-Th r-8-Orn-oxytocin (17·2 ± 2·0 times more potent that d(OEt)-oxytocin). In myometrium from pregnant women the inhibitory effects of the majority of the analogues were less pronounced. In the rat in vivo the most potent analogue 1-deamino-2-d-Trp-4-Val-8-Orn-oxytocin was 19·9 ± 2·5 times more active than d(OEt)-oxytocin. Exchanging l-tyrosine for the d form generally increased inhibitory activity as well as specificity of the analogues. Alkylation of the d-tyrosine residue did not appear to be necessary for inhibition. Substitution with d-tryptophan at position 2 gave analogues with high inhibitory potency in the rat in vitro and in vivo, but which exhibited weak effects in women in vitro. There was no correlation between the inhibitory effects on myometrium from non-pregnant and pregnant women nor between rat and human data. The high antiuterotonic activity of 1-deamino-2-d-Tyr(OEt)-4-Val-8-Orn-oxytocin and 1-deamino-2-d-Tyr(OEt)-4-Thr-8-Orn-oxytocin combined with low blood pressure and antidiuretic effects make these two analogues interesting for clinical studies. J. Endocr. (1986) 111, 125–131

scholarly journals Altered Drug Binding to Serum Proteins in Pregnant Women: Therapeutic Relevance

1981 ◽  
Vol 74 (6) ◽  
pp. 422-426 ◽  
Author(s):  
E Peruccam ◽  
M Ruprah ◽  
A Richens
Keyword(s):  
Valproic Acid ◽  
Pregnant Women ◽  
Serum Proteins ◽  
Drug Binding ◽  
Albumin Concentration ◽  
Serum Albumin Concentration ◽  
Partial Explanation ◽  
Unbound Fraction ◽  
Drug Levels

The binding of diazepam, phenytoin and valproic acid to serum proteins in vitro has been compared in pregnant women of different gestational ages and in controls. The unbound fraction of each of the three drugs was elevated during pregnancy (particularly during the last 8 weeks) probably due, at least in part, to a fall in serum albumin concentration. These findings may provide a partial explanation for the increase in the clearance of certain drugs during pregnancy and need to be taken into account when interpreting serum drug levels in clinical practice.

THIOCYANATE ION IN SERUM AS AN INTERFERING FACTOR IN THE IN-VITRO BIOASSAY OF THYROTROPHIN

1973 ◽  
Vol 57 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. R. BOURKE ◽  
S. W. MANLEY ◽  
R. W. HAWKER
Keyword(s):  
Specific Activity ◽  
Cobalt Nitrate ◽  
Ferric Nitrate ◽  
In Vitro Bioassay ◽  
Rat Serum ◽  
Thiocyanate Ion ◽  
Serum Thiocyanate ◽  
In Vitro Bioassays

SUMMARY The presence of thiocyanate ion in human and rat serum has been shown to account entirely for the non-specific activity of sera in an in-vitro bioassay for thyrotrophin. Thiocyanate was identified by its chromatographic behaviour on Sephadex G-10, G-15 and G-25, and by the ferric nitrate and cobalt nitrate tests. Cigarette smoking increased mean serum thiocyanate levels (as NaSCN) from 0·2 to 0·56 mg/100 ml. It is suggested that serum thiocyanate levels are sufficient to inhibit significantly iodide trapping in vivo and that these findings may be relevant to the non-specific responses observed with other in-vitro bioassays based on radioiodine dynamics.

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