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Biomedical subjects

V M Doctor

Publications and source records attributed to V M Doctor.

65 records · Page 4Linked to original sources
PubMedDate not supplied

Mechanism of enhancement by fucoidan and CNBr-fibrinogen digest of the activation of glu-plasminogen by tissue plasminogen activator.

The interactions of fucoidan with human glutamic type plasminogen (Glu-Plg), porcine pancreatic elastase digested plasminogen fractions and two chain tissue plasminogen activator t-PA) were investigated using fucoidan-Sepharose affinity chromatography. The results showed a high degree of affinity between fucoidan-Sepharose and Glu-Plg or PlgK(1-3) but not with PlgK4 or mini-Plg. Fucoidan-Sepharose also showed a high affinity for t-PA, which was largely reversed by 0.002 M 6-aminohexanoic acid (6-AH). The addition of fucoidan and CNBr-fibrinogen digest (CNBr-Fbg) gave the highest enhancement of the in vitro activation of Glu-Plg by t-PA in the presence of 0.002 M 6-AH. The results of affinity chromatography and enhancement studies suggested a template mechanism, since increasing the concentrations of any one of the two cofactors reversed the enhancement. Enzyme kinetic studies, using double reciprocal plots, showed that the addition of fucoidan-6-AH increased Kcat by 7-fold without affecting Km and addition of CNBr-Fbg lowered Km by 5-fold without significantly affecting Kcat while addition of the two cofactors lowered Km by 16-fold without significantly affecting Kcat. The enhancement by fucoidan-6-AH or by CNBr-Fbg of the in vitro activation of Glu-Plg by t-PA was reversed by plasminogen activator inhibitor 1 (PAI-1). Fucoidan-Sepharose affinity chromatography revealed that the binding of PAI-1 with fucoidan may be responsible for the reversal of the enhancement by fucoidan-6-AH.

Chromatography, Affinity↗
PubMedDate not supplied

Ionic modulation of the effects of heparin and 6-aminohexanoic acid on plasminogen activation by streptokinase: the role of ionic strength, divalent cations and chloride.

Studies were conducted on the effect of heparin or 6-aminohexanoic acid (6-AH) on the activation of glutamic plasminogen (Glu-Plg) by streptokinase in the presence of different concentrations of buffer, NaCl and divalent cations. Heparin and 6-AH inhibited streptokinase-mediated activation of Glu-Plg using 10 mM Tris-HCl buffer pH 7.4. This inhibition was partially reversed by the addition of 0.2-1.0 mM of Mg ions. Increasing the ionic strength of Tris-HCl buffer from 10 to 50 mM or addition of 50-150 mM of NaCl to 50 mM Tris-HCl pH 7.4 inhibited the activation of Glu-Plg by streptokinase while decreasing the % inhibition by heparin over the control samples. Double reciprocal plot of the activation of Glu-Plg by streptokinase using 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl showed that the addition of heparin lowered Vmax by 50% without affecting Km. To determine whether the inhibitory effect of heparin was specifically directed towards Glu-Plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of heparin over the controls were plotted against the inverse of the volume fraction of Glu-Plg or streptokinase after serial dilutions. The results indicated that the dilutions of streptokinase but not of Glu-Plg influenced the ratios, suggesting an interaction of heparin with streptokinase. Addition of 6-AH reversed the inhibitory effect of NaCl on the activation of Glu-Plg by streptokinase and the results of the near UV CD spectra of Glu-Plg showed that addition of 6-AH enhanced the spectra in this region with an increase in the ellipticity which was not affected by addition of NaCl.

Aminocaproic Acid↗
PubMedDate not supplied

Mechanism of the stimulatory effect of native fucoidan, highly sulfated fucoidan and heparin on plasminogen activation by tissue plasminogen activator: the role of chloride.

Native Fucoidan and unfractionated heparin enhanced by 6-fold the in vitro activation of human glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05M Tris buffer pH 7.4, while sulfated fucoidan inhibited the activation under these conditions. Double reciprocal plots of these interactions showed that sulfated fucoidan inhibited the activation in a noncompetitive manner while the enhancements by heparin or native fucoidan were due to an increase of Vmax without affecting Km. To determine whether the stimulatory effect of the individual cofactor was due to its interaction with Glu-Plg or with t-PA, experiments were performed at a fixed level of the cofactor and either varying in a serial fashion the concentration of Glu-Plg or of t-PA. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of Glu-Plg or of t-PA. The results showed that heparin interacted with Glu-Plg while native fucoidan and sulfated fucoidan interacted with t-PA. Studies were also conducted on the effect of the two fucoidans and heparin on the activation of Glu-Plg by t-PA using 0.05M Tris buffer pH 7.4 containing 0.1 M NaCl. Under these conditions, sulfated fucoidan was most effective in enhancing the activation followed by native fucoidan and heparin respectively. The results of this study showed that in presence of the buffer containing 0.1 M NaCl, heparin was interacting with t-PA while the two fucoidans were interacting with both t-PA and Glu-Plg. A comparison of the double reciprocal plots of the rate of enhancement by the cofactors using 0.05M Tris buffer pH 7.4 containing 0.1M NaCl or in presence of buffer alone showed that the cofactors were more effective using 0.05M Tris buffer pH 7.4 alone and that addition of NaCl to the buffer slowed down the reactions by decreasing Vmax without affecting Km. Circular Dichroism (CD) studies of Glu-Plg in the far ultraviolet (UV) range showed that addition of NaCl destabilized the beta sheet structure which was reversed by addition of 6-aminohexanoic acid (6-AH) or one of the cofactors, while the near UV CD spectra of Glu-Plg in presence of 0.1 M NaCl was enhanced by the cofactors by increasing its ellipticity as reported earlier for 6-AH.

Chlorides↗
PubMedDate not supplied

Effect of sulfated xylans during the interaction of [125I]-thrombin with antithrombin III or heparin cofactor II of human plasma.

[125I]-Labeled thrombin was incubated with human plasma and its interactions with the two plasma protease inhibitors antithrombin III (AT-III) or heparin cofactor II (HC-II) were investigated in the presence of oat spelts xylan sulfate (OSXS), sodium pentosan polysulfate (SP-54), and the results were compared with heparin and dermatan sulfate. Addition of OSXS or SP-54 enhanced the complexation of thrombin with HC-II or with both AT-III and HC-II depending upon the concentration and the duration of the interactions of the sulfated compounds with plasma. During the 30 s interaction, OSXS and SP-54 enhanced both AT-III-thrombin and HC-II-thrombin interaction while heparin was more selective and enhanced only the AT-III-thrombin interaction. However after a 2 min interaction, heparin showed an increase in the HC-II-thrombin interaction at higher concentrations. The complexations of AT-III-thrombin and HC-II-thrombin were reversed in the presence of 200 micrograms/ml of SP-54 during a 30 s interaction or in presence of 100 micrograms/ml of OSXS during a 2 min interaction. The Western blotting method of detecting thrombin showed that during the 10 s interaction, heparin enhanced the thrombin-AT-III complex formation while OSXS enhanced the thrombin-HC-II complex formation.

Antithrombin III↗
PubMedDate not supplied

Mechanism of the stimulatory effect of 6-aminohexanoic acid on plasminogen activation by streptokinase or tissue plasminogen activator: the role of chloride.

Studies were conducted on the mechanism of the stimulatory effect of 6-aminohexanoic acid (6-AH) during the in vitro activation of human glutamic plasminogen (Glu-Plg) by streptokinase or by tissue plasminogen activator (t-PA) and the possible role of the addition of physiological concentrations of NaCl to the buffer solution. Enhancement by 6-AH was investigated by measuring the rate of plasmin generation using chromogenic substrate H-D-glu-phe-lys-pNA (S-2403). Control studies using plasmin showed that the addition of 6-AH at concentrations below 20 mM did not significantly affect the initial rate of the amidolytic activity of plasmin with or without the addition of NaCl to 0.05 M Tris buffer (pH 7.4). On the other hand, addition of NaCl to the buffer slowed down the initial rate of activation of Glu-Plg by streptokinase or by t-PA while increasing the percent enhancement by 6-AH when compared with the controls. The ratios of the initial rates of plasmin generation in the presence or in the absence of 6-AH were plotted against the inverse of the volume fraction of Glu-Plg, streptokinase or t-PA after serial dilutions. The results showed that when the activation reactions were performed in 50 mM of Tris buffer (pH 7.4), the enhancements by 6-AH were related to its interaction with streptokinase or t-PA, while using the same Tris buffer containing 0.6 % NaCl, the enhancements by 6-AH were related to its interaction with both Glu-Plg and streptokinase or t-PA. However, upon increasing the NaCl to 0.9%, the results showed that the enhancements by 6-AH of the activation of Glu-Plg by streptokinase or t-PA were related to its interaction with Glu-Plg. The results suggested that changes in the concentrations of NaCl play a regulatory role during the activation process.

Aminocaproic Acid↗
PubMedDate not supplied

Expression of cell surface glycoprotein CD44 and integrins in breast cancers among Indian women.

Parsis, the sole surviving group of followers of Zoroaster who are settled in Bombay, have a fourfold higher incidence of breast cancer than the general population of Greater Bombay. CD44 expression was studied by immunohistochemistry in breast cancers of 50 non-Parsi and 35 Parsi women, 10 normal breast tissues, 10 proliferative lesions and 49 tissues adjoining a tumor mass. Alpha2 and beta1 integrins could be studied in only 42 malignant cases and five normal tissues. The immunohistochemistry results were correlated with other parameters including tumor grade and size, estrogen and progesterone receptor status, lymph node involvement and mitotic index. CD44 was not expressed in normal areas. Benign areas and tissues adjacent to tumor masses showed increased staining. Both Parsi and non-Parsi women showed significantly high CD44 expression. All Parsi ILCs were strongly positive for CD44. In both groups ER negativity was associated with strong CD44 positivity, while mitotic counts correlated with decreased CD44 expression in Parsis but not in non-Parsis. Alpha2 and beta1 integrins were strongly expressed on the basolateral surface of normal epithelium. However, they were downregulated in tumors. In general breast tumor tissues from Parsi and non-Parsi patients did not differ significantly with respect to most parameters. However, among Parsis lymph node involvement and CD44 correlated weakly whereas the mitotic index was inversely correlated with CD44. The reverse was true for non-Parsis. The deviation from the general pattern needs further study based on a large number of samples and appropriate use of splice variants.

Breast↗