The effect of heparin removal in vitro on the activity of tissue factor pathway inhibitor.
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Anticoagulants↗
Biomedical subjects
M Mukherjee
Publications and source records attributed to M Mukherjee.
(E)-2-[2-(1-Naphthyl)vinyl]-3-tosyl-2,3-dihydro-1,3-benzothiazole.
The title compound, C(26)H(21)NO(2)S(2), which consists of a benzothiazole skeleton with alpha-naphthylvinyl and tosyl groups at positions 2 and 3, respectively, was prepared by palladium-copper-catalyzed heteroannulation. The E configuration of the molecule about the vinyl C=C bond is established by the benzothiazole-naphthyl C-C-C-C torsion angle of 177.5 (4) degrees. The five-membered heterocyclic ring adopts an envelope conformation with the Csp(3) atom 0.380 (6) A from the C(2)NS plane. The two S-C [1.751 (4) and 1.838 (4) A] and two N-C [1.426 (5) and 1.482 (5) A] bond lengths in the thiazole ring differ significantly.
Benzothiazoles↗
Bis
In the title compound, [Zn(C(8)H(10)N(3)O(2))(2)], the Zn atom displays a highly distorted octahedral coordination involving O and N atoms of two bidentate planar ligands approximately orthogonal to each other; the dihedral angle between the ligand planes is 84.95 (4) degrees. The ligand molecules show great asymmetry in their bonding to the Zn(2+) ion, with Zn-O bond distances ranging between 2.056 (2) and 2.534 (2) A. The planar phenyl ring and the trigonal-planar geometry about the triazene N atom bonded to the phenyl ring suggest a resonance interaction extending over adjacent atoms.
Journal Article↗
A direct-method ab initio phasing of a protein, pseudoazurin, at 1.55 A resolution.
The direct-method program MULTAN88 has been applied to solve a known protein, pseudoazurin, space group P6(5), unit-cell parameters a = b = 50.0 (1), c = 98.5 (3) A, with 917 protein atoms, a Cu atom and 93 solvent water molecules in the asymmetric unit (>6,000 non-H atoms in the unit cell) and data at 1.55 A resolution. One of several trials with sets of initially random phases yielded phase estimates for 1,000 reflections with a mean phase error of 68.3 degrees that was recognized as the best available solution by the figure of merit being used. Phase extension to 2,000 largest Es showed a distorted tetrahedral geometry around the Cu site. Density modification was applied to improve the phases and the quality of the maps, starting with phases calculated from the atomic positions indicated by the first map.
Azurin↗
HMG-I/Y, a new c-Myc target gene and potential oncogene.
The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc-Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc, increased expression of HMG-I protein leads to the neoplastic transformation of both Rat 1a fibroblasts and CB33 cells. In addition, Rat 1a cells overexpressing HMG-I protein form tumors in nude mice. Decreasing HMG-I/Y proteins using an antisense construct abrogates transformation in Burkitt's lymphoma cells. These findings indicate that HMG-I/Y is a c-Myc target gene involved in neoplastic transformation and a member of a new class of potential oncogenes.
Animals↗
Anticoagulant versus amidolytic activity of tissue factor pathway inhibitor in coronary artery disease.
In view of raised levels of endothelial markers in coronary artery disease (CAD), the aim of the present study was to investigate the status of tissue factor pathway inhibitor (TFPI), another endothelium-associated glycoprotein and coagulation protease inhibitor, in CAD. The intravascular pool of TFPI is heterogeneous with respect to assay-dependent activity. While the standard amidolytic assay works well with both full-length and truncated (lipoprotein-associated) TFPI, the anticoagulant assay works better with the former. The anticoagulant activity of TFPI can be estimated using dilute tissue factor (TF) to trigger clotting of plasma. In the present study, recombinant TF diluted 2,000-fold was used to initiate coagulation. A dose-dependent shortening of clotting time of normal plasma pools with polyclonal antibody against the C-terminal but not the N-terminal peptide of TFPI demonstrated the importance of the C-terminal region, and hence that of full-length TFPI, in conferring its anticoagulant activity, corroborating current opinion. As a further confirmation, the C-terminal peptide itself prolonged dilute TF clotting time of normal pooled plasma in a concentration-dependent manner. The amidolytic and anticoagulant activities of TFPI were determined in 20 patients with clinically and angiographically assessed CAD and in 68 asymptomatic controls. The mean +/- SD ages of patients and controls were 54.9 +/- 10.3 and 48.8 +/- 11.6 years, respectively, the difference being statistically significant (P = 0.04). The mean TFPI activity measured by amidolytic assay was comparable for patients and controls (1.2 +/- 0.3 and 1.3 +/- 0.5 U/ml, respectively). However, the dilute TF clotting time was 115 +/- 26 s in patients, against 99 +/- 10 s in controls (P < 0.0001, irrespective of age adjustment). Since none of the patients had received heparin or had coagulation factor deficiency that may interfere with the assay, prolongation of clotting time may be attributed to the presence of TFPI, particularly the full-length form. To verify this inference, 33 extra aliquots left over from 88 samples (62.5%), 21 from controls and 12 from patients, were incubated with 1:10 diluted antibody against the C-terminal peptide of TFPI prior to dilute TF assay. The mean clotting time of both patients and controls decreased, and the between-group difference leveled (90 +/- 10 versus 88 +/- 20 s for controls and patients, respectively; P = 0.841). The mean drop in clotting time was 9% for the controls and 24% for the patients. This illustrates the specificity of dilute TF assay for full-length TFPI and supports the conclusion that relative to lipoprotein-associated TFPI, the proportion of the full-length form was possibly greater in patients with CAD. Contribution of lipoprotein-associated TFPI to the overall anticoagulant activity by its activated factor X-dependent inhibition of activated factor VII-TF complex seems less important considering the similar between-group mean amidolytic activities.
Anticoagulants↗
Role of dopamine in the modulation of adrenal gland responses in the osmotically stressed pigeons, Columba livia.
Salt loading on pigeons (C. livia) had stimulatory effects on brain amines (dopamine and 5-hydroxytryptamine), corticosterone, norepinephrine and epinephrine contents of adrenal gland. Conjoint administration of dopamine with hypertonic saline restored the brain amines and corticosterone of adrenal gland, but had no effect on catecholamine (CAM) contents of adrenal medulla. The excessive release of CAM in the plasma indicates sympathetic stimulation after both the treatments.
Adrenal Glands↗
On the application of phase relationships to complex structures. XXXVI: some experiments with a small protein without heavy atoms.
The direct-methods program MULTAN88 has been applied to a known protein, ribonuclease (RNAP1), containing 808 non-H atoms, including five S atoms, plus 83 ordered solvent water molecules. Phase sets with mean phase errors between 69 and 75 degrees were selected by modified figures of merit for trials with the full data at 1.17 A resolution and also with restricted data at 1.25 and 1.5 A resolution. These figures of merit had previously only been applied to protein structures containing heavy atoms, and this is the first demonstration of their usefulness with no heavy atom present. An initial set of 1091 phases from a 1.17 A trial was developed by an objective procedure to give the full structure with a residual of 0. 21, which agrees well with the published structure.
Crystallography, X-Ray↗
Haemodynamic responses and changes of haemostatic risk factors in cold-adapted humans.
Epidemiological studies have shown an increase in acute myocardial infarctions or deaths due to myocardial infarction in colder weather; the mechanisms most likely involve increased blood levels of haemostatic risk factors, and increases in arterial blood pressure and heart rate. We studied the relationship between cold adaptation, haemostatic risk factors and haemodynamic variables. Cold adaptation was obtained by a programme of immersion of the whole body up to the neck in a water-filled bath, the temperature of which was gradually decreased from 22 degrees C to 14 degrees C, time of exposure being increased from 5 to 20 min over a period of 90 days. We studied 428 patients (44% men) and measured blood levels of fibrinogen, plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator antigen (t-PA), plasma viscosity, von Willebrand factor, D-dimer and platelet count, both at baseline and after 90 days of daily immersion. There were significant reductions in von Willebrand factor (-3%; p < 0.001), and plasma viscosity (-3.0 s; p < 0.001), and a mild but significant increase in PAI-1 (+0.3 IU/ml; p = 0.02). The pressure rate product (systolic blood pressure x heart rate) was also significantly lower after cold adaptation (-310; p = 0.004). Cold adaptation, compared with exposure to cold weather, induces different haemodynamic responses and changes of blood levels of haemostatic risk factors.
Acclimatization↗
Ab initio structure determination of a small protein, rubredoxin, by direct methods.
The direct-methods program SAYTAN has been applied successfully to a known protein, rubredoxin, which contains 52 amino-acid residues including an FeS4 unit, a sulfate ion and 102 solvent water molecules. Starting with initially random phases, useful sets can be obtained from multiple trials and selected by figures of merit at different resolutions. Phase extension followed by weighted Fourier recycling reveals a recognizable structure of rubredoxin. The model is refined against 1 A resolution data to an R factor of 14.5% using the program SHELXL93.
Bacterial Proteins↗
A comparative study on the Mitsuda type response to antigens of chemoautotrophic nocardioform bacteria and to standard lepromin in leprosy patients.
Anergy, or contrarily, Mitsuda-type responses towards 4 chemoautotrophic nocardioform antigens (CAN-Ags) and a control standard lepromin were tested in 73 LL, TT and borderline cases of leprosy. The antigens injected per patient varied from a maximum of 5 to a minimum of 2. Complete anergy to CAN-Ags was seen in 92/92 instances tested on 24 LL cases. The anergy was weakly modified or unmodified in 3 other LL cases which had been vaccinated before. Concurrent studies with the same antigens tested on 33 TT cases showed clear-cut, dose-dependent, Mitsuda-type late responses in 80/81 instances. The CAN bacteria, therefore, despite their origin from different unrelated leprous human, mouse footpad (MFP) and armadillo tissues, appeared to be identical with each other and also probably related to the leprosy bacillus, on the basis of these parameters.
Actinomycetales↗
Enhancement of lipoprotein lipase activity by tissue factor pathway inhibitor.
The effect of a basic synthetic peptide, representing the C-terminal region of tissue factor pathway inhibitor (TFPI - Lys254 - Met276), as well as that of the whole protein, on the activity of lipoprotein lipase (LPL) is described. The activity of bovine LPL was measured by chromogenic assay using a water-soluble chromogenic substrate, p-nitrophenyl butyrate. Five and 10 microM concentrations of the peptide increased Vmax of bovine LPL by 48.9% and 85.6% respectively as compared with the buffer control without affecting Km. Poly l-lysine, though positively charged did not have any effect, suggesting the importance of the amino acid sequence of the test peptide. On the other hand, 0.25, 0.5 and 1.0 mM n-butyric acid - a product of LPL catalysis in the chromogenic assay, when added to the incubation mixture decreased Vmax non competitively by 22.8%, 40.4% and 63% respectively as compared with buffer control, confirming the known product inhibition of LPL. A 100-fold molar excess of n-butyric acid produced inhibition of the LPL reaction as compared with the synthetic peptide which produced potentiation, suggesting a 1:100 stoichiometric interaction of the peptide with n-butyric acid. At a fixed concentration of 0.25 mM substrate, 10 nM full length recombinant TFPI, containing the basic C-terminal domain, increased velocity of LPL reaction by 39.4% as compared with buffer control. The same concentration of two-domain recombinant TFPI (TFPI1-160) had no effect. It is possible that negatively charged n-butyric acid is sequestered by the positively charged peptide or the basic region of recombinant full length TFPI. Relieving of product inhibition could then be a possible mechanism of the observed potentiation of bovine LPL activity by the basic peptide or full length recombinant TFPI. The 39.4% increase in reaction velocity of LPL catalysis produced by 10 nM full length recombinant TFPI was comparable to 38.9% increase produced by 5 microM of the basic peptide under the same conditions. A further increase of 78.7% was brought about by 10 microM concentration of the same peptide. The reason for about 500-fold increase in the potency of the whole protein as compared with that of the peptide is not clear. It is possible that in its tertiary conformational state, the whole protein is able to sequester product and relieve product inhibition more effectively than the short linear peptide. Rabbit polyclonal antiserum against the basic peptide partially inhibited LPL activity of human post heparin plasma, measured by radioenzymatic assay using triolein substrate. Since post heparin plasma contains full length TFPI, binding of the added antibody to its basic C-terminus and hence the relative unavailability of latter for product sequestration (oleic acid in this case) could explain the observed inhibition of human LPL activity by antibody against the peptide. Thus by enhancing lipase activity, full length TFPI may facilitate hydrolysis of triglyceride and concomitantly lower factor VII coagulant activity as demonstrated earlier, particularly after heparin injection when both TFPI and LPL are released in circulation.
Amino Acid Sequence↗
Development of in vitro screening system for assessment of antifilarial activity of compounds.
Evaluation of antifilarial activity of new potential agents in vivo is extremely time consuming and uneconomic. In the present study effort has been made to develop an in vitro screening method using Acanthocheilonema viteae, a subcutaneously dwelling rodent filariid with anaerobic metabolic characteristics like human filariids, W. Bancrofti/Brugia malayi as test parasite. Motility test and tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) based colorimetric assay were used as parameters in in vitro assay. Results showed that 92.3% of compounds (in vivo active) could be picked up in the in vitro assay when both adults and microfilarae (mf) were used simultaneously. Mf and adult stages separately detected, respectively, 84.6 and 69.2% of in vivo active compounds. The adults and mf separately and both the life stages together exhibited, respectively, 80.0, 50.0 and 80.0% false positive results in the in vitro test with in vivo inactive compounds. It is felt that mf stage when used in in vitro test using motility and MTT assays as parameters would be useful in primary screening of new potential filaricides.
Animals↗
Unexpected electrophoretic migration of RNA with different 3' termini causes a RNA sizing ambiguity that can be resolved using nuclease P1-generated sequencing ladders.
It has been widely believed that the electrophoretic migration difference of otherwise identical RNAs with a P versus OH terminus would be the same as occurs for DNA, a fairly reproducible approximately 1/2 nucleotide (nt) offset. RNA with a 5'-OH indeed migrates </=1 nt slower than if it had a 5'-P. Surprisingly, however, RNA with a 3'-OH terminus (generated by many cellular RNases of interest) migrates anywhere from approximately 1/4 to approximately 2 nts slower than the otherwise identical molecule with a 3'-P or 2', 3'-cyclic-P terminus (as present on standard RNase-generated sequencing ladders). This previously unrecognized variability in electrophoretic migration offset causes a 1-2 nt ambiguity in a commonly used method of RNA size determination. We also show two ways to overcome this problem and enable rigorous sizing of 3'-OH terminating RNAs. Most convenient is to use sequencing standards generated by nuclease P1, which is generally sequence-nonspecific but we show becomes G-specific or A-preferential under certain reaction conditions.
Animals↗
Cellular immune response of Mastomys and gerbils in experimental filariasis.
OBJECTIVE: To determine mitogenic and antigen-specific cellular immune responses of two species of rodents, viz. Meriones unguiculatus and Mastomys coucha to assess the usefulness of the A. viteae/Mastomys model for cellular immune studies in experimental filariasis. METHODS: Lymphocyte blast transformation test (LTT) using spleen cells of normal and A. viteae infected animals. RESULTS: The proliferative response of gerbils was much higher than that of Mastomys to both ConA and filarial antigens. Cells of both species of rodents did not respond to microfilarial (mf) antigen, however, their mitogenic response differed during infection. Some degree of nonspecific suppression was observed in gerbils during prepatent and patent stages of infection, while Mastomys revealed highest proliferation during patent microfilaraemia. Mastomys cells did not respond to adult or mf antigen, while adult-specific proliferation was detected in the case of gerbils. CONCLUSION: The A. viteae/gerbil model shows more similarity to human filarial infection regarding cellular immune response. Markedly low responsiveness of a high percentage of Mastomys and wide variations in the cellular response to nonspecific mitogen limit the usefulness of Mastomys coucha in immunological studies, especially cellular immunity.
Animals↗
Chronic fatigue syndrome: physical and cardiovascular deconditioning.
We investigated whether chronic fatigue syndrome (CFS) patients have physical and/or cardiovascular de-conditioning, in 273 CFS patients and 72 healthy controls. We used laboratory tests to assess haematological, biochemical, endocrinological and immunological systems. The cardiovascular system was assessed by echocardiography and carotid echography. Body composition was determined by dual energy X-ray absorptiometry (DEXA). CFS patients had smaller left ventricular end systolic (p < 0.001) and diastolic (p = 0.008) dimensions but thinner posterior walls (p = 0.02) than corresponding values in healthy controls. Left ventricular mass was also reduced in CFS patients (p = 0.006). Both maximum (p < 0.001) and minimum (p < 0.008) diameter of the carotid artery were smaller in CFS patients. The laboratory screening tests showed significant differences in serum albumin (p = 0.05), phosphate (p = 0.02), HDL-cholesterol (p = 0.03), HDL:total cholesterol ratio (p = 0.01), triglycerides (p = 0.02), neutrophils (p = 0.01) and thyroid-stimulating hormone (p = 0.04) between CFS patients and controls. Male CFS patients had an increased percentage of fat mass compared with healthy male subjects (p = 0.02). This large group of CFS patients had evidence of physical and cardiovascular de-conditioning, suggesting that in these patients a graded exercise programme could lead to physical reconditioning and could increase their ability to perform physical activities.
Adult↗
Association of DEXA-assessed total body fat mass with serum lipids and haemostatic variables.
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Absorptiometry, Photon↗
Thrombogenicity of heparin and non-heparin bound arterial prostheses: an in vitro evaluation.
The effect on graft thrombogenicity of binding heparin to the luminal surface of prosthetic arterial grafts was investigated. Venous blood was obtained from healthy volunteers and exposed for 30 minutes to tubular segments of standard knitted dacron, polytetrafluoroethylene (PTFE) and a recently introduced heparin-bound knitted dacron graft. After this exposure the fibrinogen level of each sample was measured. The median (range) fibrinogen levels (expressed as a percentage of that in unexposed blood samples) were: standard dacron 3.5% (0-5.4%); PTFE 95.5% (0-121.1%); and heparin-bound dacron 79.8% (3.8-109.6%). Fibrinogen levels in the standard dacron group were significantly less than that of the PTFE and heparin-bound dacron groups (P < 0.05). No significant difference was found between the fibrinogen levels of the PTFE and heparin-bound dacron groups (P = 0.35). These findings suggest that heparin binding significantly reduces fibrinogen consumption and hence may reduce graft thrombogenicity.
Anticoagulants↗