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A Electricwala

Publications and source records attributed to A Electricwala.

At least 19 recordsLinked to original sources
PubMed1994

Isolation of prolactin from human pituitary glands: some safety aspects of an improved partial purification procedure.

Human prolactin was partially purified from pituitary glands by a combination of alkaline extraction, ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration. The early steps in the purification were carried out under stringent safety conditions to minimise the risk of exposure to potential viral pathogens present in human tissue. Alkaline extraction was carried out over a short time interval to limit possible deamidation of prolactin without loss in recovery. During chromatography on phenyl-Sepharose, it was found that bound prolactin could be eluted with aqueous buffer, in contrast to the organic solvents used by previous investigators. Subsequent gel filtration removed high molecular weight contaminant proteins and further confirmed the monomeric form of purified prolactin. This material could be further purified to near homogeneity by ion-exchange chromatography and other separation techniques. Some guidelines on the safety precautions undertaken during the extraction of prolactin are described.

Biotechnology↗
PubMed1993

The complete amino acid sequence of a hirudin variant from the leech Hirudinaria manillensis.

Unlike the European leech Hirudo medicinalis, the Asian jawed leech Hirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions of Hirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as with Hirudo leech, various isoforms of hirudin also exist in Hirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

Amino Acid Sequence↗
PubMed1992

Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis.

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.

Amino Acid Sequence↗
PubMed1991

Isolation of thrombin inhibitor from the leech Hirudinaria manillensis.

The leech Hirudinaria manillensis belongs to the same family as the medicinal leech Hirudo medicinalis, which has been widely used for the study of hirudin, a specific thrombin inhibitor. A similar inhibitor has now been isolated from the heads of the Hirudinaria leech by acetone/acid extraction and further purified to near homogeneity by ion exchange chromatography followed by affinity chromatography on thrombin-agarose and reverse phase HPLC. The purified material was recovered at about 10-15% yield and had a specific activity of about 12,000-14,000 ATU/mg, similar to other hirudin variants. The inhibitor was shown to be homogenous by sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 8 M urea with an apparent molecular mass of about 7000 daltons under reducing conditions. Comparison of the anticoagulant effect on human plasma by partial thromboplastin time assay have shown that the inhibitor from Hirudinaria has similar potency as hirudin variant 1 at equivalent dosage. However, it does not cross-react with monoclonal antibodies towards recombinant hirudin variant 1. Comparison of the N-terminal amino acid sequence up to residue 25 also indicates differences at positions 2, 13, 17 and 24 between the two thrombin inhibitors. These findings indicate that the primary anticoagulant present in the leech Hirudinaria is a potent thrombin inhibitor (Bufrudin) with biological activity similar to hirudin, but differs in its structural and immunological properties.

Amino Acid Sequence↗
PubMed1990

Physico-chemical properties of recombinant desulphatohirudin.

Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.

Chemical Phenomena↗
PubMed1990

Effect of hirudin on tissue plasminogen activator induced clot lysis.

The effect of recombinant hirudin in the in vitro tPA fibrinolytic and thrombolytic activity was investigated. The activity was evaluated by following lysis of radiolabelled fibrin or plasma clot formed in the presence of tPA alone or with hirudin. The results obtained indicate that increasing concentrations of hirudin had a potentiating effect, with faster clot lysis rates and reduced time to complete lysis. However, when radiolabelled plasma or whole-blood clots were immersed in autologous plasma in the presence of tPA and hirudin, no significant difference in the lysis rates and time to complete lysis was observed. The findings suggest that hirudin or hirudin-thrombin complex interferes with the forming fibrin, thereby making clots more susceptible to lysis, while the presence of hirudin in the surrounding medium during lysis of formed clots helps to rapidly neutralize active thrombin released during clot lysis, thereby preventing further activation of coagulation. Thus, use of hirudin as an anticoagulant during thrombolytic therapy may prove to be helpful in reducing the incidence of reocclusion.

Fibrinolysis↗
PubMed1989

Lectin affinity bioassay: an assay method for glycoprotein enzyme.

A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.

Alkaline Phosphatase↗
PubMed1989

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines.

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.

Azacitidine↗
PubMed1987

Amplification of tissue plasminogen activator expression from epithelial cell lines.

Two established non-malignant epithelial cell lines, one derived from human breast tissue (BEB), the other from guinea pig ear keratocytes (GPK), have been shown to secrete a tissue plasminogen activator (tPA). The protein yield of this epithelial enzyme is similar to that from the malignant cell line, Bowes melanoma, but as the specific activity is approximately 10-20 fold lower, means of potentiating the yield are being sought. The enzyme is mainly expressed during the cell growth phase rather than from stationary culture cells. Also, production has to be a 2-step operation with initial growth to about 70% confluency in the presence of serum followed by a change to serum-free conditions for the final period of growth after which the enzyme is harvested. To increase the enzyme yield, means of amplifying enzyme expression, of establishing a serum-free medium so that the enzyme can be continuously harvested, and stimulation of stationary-phase cells have been studied. Of the many regulatory agents studied only the hypomethylating agent 5-azacytidine brought about significant increases (3-5 fold) in enzyme secretion. The effect of a number of mitogenic lectins was also investigated and resulted in further increases of enzyme yield (15-20 fold). Concanavalin A considerably extended the culture period in which enzyme secretion occurred and the reasons for this were investigated using tritiated thymidine.

Animals↗
PubMed1986

Stimulation of tissue plasminogen activator production from epithelial cell lines.

The aim of this study was to investigate the possibility of enhancing the yield of tissue plasminogen activator (tPA) from two epithelial cell lines of normal (non-malignant) derivation grown in tissue culture. The three agents used in this investigation were chosen because of their proven enhancing effect on analogous cells or products. The anabolic hormone stanozolol was found to have no significant stimulatory effect on these cell lines. A phorbol acetate (12-O-tetradecanoylphorbol 13-acetate) caused a twofold enhancement in tPA yield but the most significant results were obtained with 5-azacytidine. This agent increased the yield by up to fourfold in small stationary cultures and threefold in large-scale microcarrier cultures. A combination of azacytidine and phorbol acetate did not have an additive effect on total yield but did alter the kinetics of tPA expression with time. Indications were that the maximum yield with these types of potentiating agents was achieved as it could not be increased by using a combination of two different agents.

Animals↗
PubMed1986

Evaluation of epithelial tissue plasminogen activator as a thrombolytic agent in a rabbit model of venous thrombosis.

The thrombolytic effect of guinea pig keratocyte plasminogen activator was evaluated in rabbits with experimental jugular vein thrombosis and compared with human melanoma activator. Both the activators were infused locally at two dose levels in groups of three rabbits. Infusion of 6,000 and 16,000 I.U./rabbit over 4 hr resulted in 43 and 62% lysis with guinea pig keratocyte activator whereas melanoma activator induced 59 and 66% lysis with 6,000 and 12,000 I.U./rabbit. Thrombolysis with both these activators was not associated with systemic activation of the fibrinolytic system or fibrinogen breakdown. It is concluded that the guinea pig keratocyte activator is a specific thrombolytic agent with an in vivo potency very similar to the melanoma activator.

Animals↗
PubMed1985

In vitro studies on the fibrinolytic, thrombolytic and fibrinogenolytic properties of a tissue plasminogen activator from guinea pig keratocytes.

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37 degrees C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (less than 50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3-6 hr with the concentration of GPK activator in the range of 1-5 micrograms/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

Animals↗
PubMed1985

Purification and properties of plasminogen activators from epithelial cells.

Two epithelial plasminogen activators were purified from the serum-free conditioned medium of guinea pig keratocytes (GPK) and human breast epithelial (BEB) cells in culture. The cells were cultured on Cytodex 3 microcarrier beads in Eagles' minimum essential medium. The purification procedure was essentially as described by Rijken and Collen (1981) [J. Biol. Chem. 265, 7035-7041]. The specific activities of the purified GPK and BEB activators were 12500 and 6000 IU/mg. Unlike other tissue activators, both the epithelial activators had an isoelectric point of approximately 4.7 +/- 0.2. Pure enzymes were shown to be homogeneous by dodecyl sulphate/polyacrylamide gel electrophoresis with an apparent molecular mass of 62 +/- 2 kDa under reducing conditions. Immunological experiments have shown that both the activators are different from urokinase and do not cross react with anti-urokinase antibodies. Both GPK and BEB activators bound tightly to fibrin clots in vitro. Preliminary N-terminal sequence results indicate that both the epithelial activators appear to be similar to one another but different from melanoma and other tissue activators. These findings indicate that the plasminogen activators secreted by epithelial cells represent a unique and different class of tissue plasminogen activator.

Amino Acid Sequence↗
PubMed1985

The production and properties of a tissue plasminogen activator from normal epithelial cells grown in microcarrier culture.

A plasminogen activator with different biochemical and physical properties to the one obtained from Bowes melanoma cells has been isolated from a normal epithelial cell line derived from guinea-pig keratocytes (GPK). Cell growth and enzyme production is carried out in microcarrier cultures using up to 15 g Cytodex 3 per litre. Cells are maintained in optimum conditions by the use of a closed perfusion loop and the system has been scaled up to 20 litres. Initially enzyme production was in serum-free medium following a growth phase in serum supplemented medium. However, the discovery that the cells produce the enzyme mainly during the replicative phase has led to investigating the use of serum-substitutes and growth factors on cell growth and enzyme production. The aim is to harvest the enzyme after the growth phase which precludes the use of high (greater than 1%) serum concentrations. The biochemical and biophysical properties of the enzyme are described.

Animals↗