Isolation of Borrelia burgdorferi--Thunder Bay District, Ontario.
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Animals↗
Biomedical subjects
M Banerjee
Publications and source records attributed to M Banerjee.
Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.
Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.
Animals↗
c-myc oncogene expression and cell proliferation in mixed oligo-astrocytoma.
Mixed gliomas (oligo-astrocytomas) are brain tumours with an admixture of 2 different cell populations: astrocytes and oligodendroglia. On the basis of histological features and behaviour, these tumours are classified as low-grade mixed gliomas (MG) and malignant mixed gliomas (MMG). We have studied the relationship between c-myc protein expression and cellular proliferation in this class of tumours. Using antibody c-33 for c-myc and PC-10 for the proliferating cell nuclear antigen (PCNA), immunohistochemistry was performed on 14 MG and 9 MMG. PCNA was increased in MMG as compared to MG in both astrocytic and oligodendroglial areas. However, more c-myc-positive cells were seen only in the astrocyte areas of MMG. Analysis of the relationship of c-myc and PCNA suggests that the correlation of c-myc with cellular proliferation is dependent on tissue type and differentiation status.
Antibodies, Monoclonal↗
The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini.
A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.
Bacteriolysis↗
A novel Ty1-mediated fragmentation method for native and artificial yeast chromosomes reveals that the mouse steel gene is a hotspot for Ty1 integration.
We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24-retrotransposon insertions into two different mouse-derived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into a YAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.
Animals↗
Nitric oxide and nitroglycerin reversal of pulmonary vasoconstriction induced by alpha-activation during exercise.
We have previously shown in sheep that pulmonary vascular resistance decreases rapidly after the onset of constant exercise, followed by a slower and smaller second vasodilation. The second phase is partly regulated by alpha- and beta-adrenoceptor activation. We examined the effect of inhaled nitric oxide (NO; 40 ppm) and intravenous nitroglycerin on beta-adrenergic blockade-induced pulmonary vasoconstriction during exercise. In paired studies, we exercised eight sheep at a constant rate of 4 miles per hour for 4 min on a treadmill and measured the hemodynamic response during beta-blockade (propranolol, 1 mg i.v.) with and without 40 ppm inhaled NO or continuous infusion of nitroglycerin (3.2-4.0 micrograms.kg-1.min-1). beta-Blockade resulted in a higher pulmonary vascular resistance during steady-state exercise (40-240 s) than in the unblocked state; reduction in pulmonary vascular resistance during the second phase of exercise was smaller with beta-blockade (13-16%) than with control exercise (26-30%). Inhaled NO and nitroglycerin reversed the beta-blockade-related pulmonary vasoconstriction to the levels of control exercise. Inhaled NO and intravenous nitroglycerin also reversed the pulmonary vasoconstriction produced by intravenous phenylephrine at rest. We conclude that exogenous NO, delivered by gas inhalation or via nitroso compounds, opposes and fully reverses alpha-receptor-activated pulmonary vasoconstriction during exercise in sheep.
Administration, Inhalation↗
PGF2 alpha causes bronchoconstriction and pulmonary vasoconstriction via thromboxane receptors in rat lung.
We determined the vascular and airway effects of PGF2 alpha and its mechanism of action on isolated-perfused lungs of rats were isolated and perfused at 50 ml/kg/min with Krebs-Henseleit bicarbonate buffer solution containing 3% bovine serum albumin. The lungs were ventilated with 21% O2 and 5% CO2 at a tidal volume of 2 ml. frequency of 60 per minute and positive end expiratory pressure of 3 cmH2O. Following injection of 50 micrograms PGF2 alpha into the afferent pulmonary catheter, there was a marked rise in pulmonary arterial pressure (Ppa) and in resistance to airflow across the lung (RL) and a fall in dynamic lung compliance (Cdyn). Double vascular occlusion technique revealed that 29% of the rise in Ppa was due to an increase in upstream and 71% to downstream resistance. N omega-nitro-L-arginine, 100 microns, a NO synthase inhibitor potentiated the Ppa response two-fold with significant change in airway mechanics. Rat atrial natriuretic factor (r-ANF), 40 micrograms quickly reversed the changes in Ppa, RL and Cdyn. Infusion of r-ANF prior to PGF2 alpha attenuated the Ppa response by 38%, RL by 44% and Cdyn by 12%. SQ 29548, a thromboxane receptor blocker and Cl, a protein kinase C (PKC) inhibitor, fully blocked both the vascular and airway responses to PGF2 alpha. PGF2 alpha is a constrictor of pulmonary vessels and airways in rat lungs via thromboxane SQ 29548 receptors, thansduced by intracellular PKC.
Animals↗
Infrequent alteration of the c-myc gene in human glial tumours associated with increased numbers of c-myc positive cells.
Twenty five human glial tumours of different grades of malignancy were examined by Southern blotting and polymerase chain reaction (PCR) for alterations (rearrangements, amplification and deletions) in the c-myc gene. Number of c-myc positive cells per thousand cells were also counted in all the tumours after immunohistochemical staining for c-myc protein was done on fixed sections of the tumours. No tumours exhibited any amplification of the gene, as found by Southern blotting. One astrocytoma and one mixed glioma showed some rearrangements in the 3' end of the gene, as detected by Southern blotting and hydridization. These two tumours had higher number of c-myc positive cells than in other tumours of the same histopathological groups. Deletion in the first promoter region, as determined by PCR, was seen in only one astrocytoma. However, the number of c-myc positive cells in that tumour did not show any deviation from that found in other astrocytomas. In light of present literature, it is speculated that the 3' rearrangements may be the cause of increased number of c-myc immunopositive cells in those tumours by disrupting the 3' end of the gene leading to increased c-myc mRNA stability. Such a mechanism may play a part in small subset of glial and possibly other tumours.
Base Sequence↗
Isolation of Borrelia burgdorferi, the Lyme disease spirochete, from rabbit ticks, Haemaphysalis leporispalustris--Alberta.
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Alberta↗
Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein.
A Ca(2+)-dependent sialic acid-binding protein (SABP) of human endometrium, which specifically bound to human sperm head plasma membrane in vitro, was found to increase the percentage motility and acrosome-reacted pattern of uncapacitated spermatozoa. The protein was synthesized in the endometrium and secreted into the uterine fluid. This intra-uterine factor, which is apparently advantageous in vitro in inducing human sperm capacitation, may play a significant role in promoting the post-release maturation of ejaculated spermatozoa by enhancing 45Ca uptake into spermatozoa by a pathway which is insensitive to calcium-channel blockers. However, the 45Ca uptake could be enhanced on exposure to the divalent cation ionophore A23187 and inhibited in the presence of the calmodulin inhibitor trifluoperazine. The SABP also induces an increase in intracellular Ca2+ in spermatozoa, as seen by FURA-2 AM studies. Furthermore, overlay studies show human SABP to be a Ca(2+)-binding protein. The data presented here suggest that SABP induces in-vitro sperm capacitation and the subsequent acrosome reaction by increasing intracellular Ca2+ concentration.
Agglutinins↗
Interaction of ustiloxin A with bovine brain tubulin.
Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent as inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [3H]colchicine to tubulin. The combination of ustiloxin A with cholchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.
Amino Acid Sequence↗
Purification and characterization of a sperm-binding glycoprotein from human endometrium.
A sialic-acid-binding protein (SABP) was purified to apparent homogeneity from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females. The 54 kDa monomer was found to be an O-linked glycoprotein with a total carbohydrate content of 34%. This protein agglutinated washed 2% v/v rabbit red blood cells (RBC) in the presence of calcium. Amongst sialic acids and sialoglycoproteins tested for haemagglutination inhibitory activities, N-glycolyl neuraminic acids and human alpha 1-acid glycoprotein were found to be the most potent, the agglutination activity being totally abolished on desialylation of the RBC in the presence of neuraminidase. Western blot studies showed it to be present in the uterine fluid but absent in normal female serum and in full-term placenta. It was also absent in endometrial homogenates of some cases of unexplained primary infertility. Specific binding studies and Scatchard analysis revealed that 125I-labelled human SABP ligand can bind to human spermatozoa with a Ka = 2.6 x 10(9) M-1, their receptors probably being glycoconjugates having a terminal sialic acid moiety, since the sperm-protein interaction could also be abolished when spermatozoa were desialylated with neuraminidase. The binding occurred specifically on the sperm head plasma membrane and decreased markedly when spermatozoa were previously capacitated in vitro using human serum albumin, implicating the possible loss of a sialoglycoprotein receptor to which the ligand binds during capacitation. The biological importance of this sperm-binding secretory glycoprotein and its functional significance in human reproduction have been discussed.
Adult↗
Changes in pulmonary vascular tone during exercise. Effects of nitric oxide (NO) synthase inhibition, L-arginine infusion, and NO inhalation.
Nitric oxide (NO) is a potent endogenous vasodilator. Its role in the normal and stressed pulmonary circulation is unclear. To better understand the importance of endogenous NO in normal physiological responses, we studied the effects of altered NO availability on the change in pulmonary vascular tone that accompanies exercise. In paired studies we measured blood flow and pressures in the pulmonary circulation at rest and during treadmill exercise at a speed of 4 mph with and without (a) N omega-nitro-L-arginine, 20 mg/kg intravenously, a selective inhibitor of NO synthase; (b) L-arginine, 200 mg/kg intravenously, substrate for NO synthase; (c) combination of the inhibitor and substrate; and (d) inhalation of NO > 30 ppm, to determine if endogenous release of NO elicits maximal vasodilation. In addition, we sought to determine the site of NO effect in the pulmonary circulation by preconstriction with either U-44619 or hypoxia (fraction of inspired O2 = 0.12) using a distal wedged pulmonary catheter technique. NO synthase inhibition raised pulmonary vascular tone equally at rest and exercise. L-Arginine reversed the effects of NO synthase inhibition but had no independent effect. NO inhalation did not reduce pulmonary vascular tone at rest or enhance the usual reduction in pulmonary vascular resistance with exercise. The effect of NO synthase inhibition was in pulmonary vessels upstream from small veins, suggesting that endogenous NO dilates primarily small arteries and veins at rest. We conclude that, in sheep, endogenous NO has a basal vasodilator function that persists during, but is not enhanced by, exercise.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Airway and vascular effects of 8-epi-prostaglandin F2 alpha in isolated perfused rat lung.
The effects of 8-epi-prostaglandin (PG) F2 alpha, a recently discovered noncyclooxygenase free radical-catalyzed product of arachidonic acid, on pulmonary vascular and airway tone, its potency, and its mechanism of action were studied. Progressively increasing bolus doses (1.0, 5.0, 10.0, and 20.0 micrograms) of 8-epi-PGF2 alpha were injected into the pulmonary artery catheter of 18 isolated rat lungs, and a single dose (40.0 micrograms) was injected into 7 additional rat lungs. The lungs were perfused with Krebs-Henseleit buffer solution containing 3% bovine serum albumin at 50 ml.kg-1.min-1 during ventilation with 21% O2-5% CO2-74% N2. 8-Epi-PGF2 alpha caused rapid pulmonary vascular and airway constrictor responses, which were followed by a gradual return over 10 min to baseline levels. Double vascular occlusion at peak rise in pulmonary arterial pressure (Ppa) revealed a 28% increase in arterial resistance. The rise in Ppa with 20 micrograms of 8-epi-PGF2 alpha was approximately twofold greater than with 20 micrograms of the cyclooxygenase-derived prostaglandin PGF2 alpha. The addition of 100 microM N-nitro-L-arginine, a blocker of endothelium-derived relaxing factor, in the perfusate potentiated the rise in Ppa by 244%. Injection of 40 micrograms of rat atrial natriuretic factor at peak response to 20 micrograms of 8-epi-PGF2 alpha accelerated the return to baseline Ppa, resistance to airflow across the lung, and dynamic lung compliance values.(ABSTRACT TRUNCATED AT 250 WORDS)
Air Pressure↗
Effects of rhGM-CSF on myeloid clonogenic cells in acute myelogenous leukemia patients.
The effects of rhGM-CSF in vivo on the myeloid clonogenic cells present in 6 AML patients was evaluated. The relative number of clonogenic cells fell in 4 of the 6 patients. The effects of rhGM-CSF on the percentage of clonogenic cells in S phase and the sensitivity of clonogenic cells to cytosine arabinoside varied among the patients. These effects were not related to the effects of rhGM-CSF on the white blood cell count or on the proliferative rate of the leukemia cell population as a whole.
Adult↗
Sumatriptan in the treatment of acute migraine with aura.
The efficacy of the selective 5HT1-like agonist sumatriptan in acute treatment of classical migraine (i.e. migraine with aura) was assessed in a double-blind, placebo-controlled, parallel group randomized trial. An oral dose of 200 mg was chosen on the basis of the efficacy rates achieved (70-85%) with 70-280 mg in open studies (1, 2). The dose of 200 mg was also chosen for the study because preliminary data from an oral pilot study indicated that efficacy increased with increasing dose up to 200 mg. Each patient was treated for a maximum of three separate attacks of migraine with aura within a three months' period. Three attacks were treated so that we could examine consistency of response across more than one attack. For attack 1, 200 mg sumatriptan was significantly more effective, safe and well tolerated than placebo at relieving headache 2 h after treatment was given (p = 0.023). In subsequent attacks, i.e. in attacks 2 and 3, there was no such significant effect of sumatriptan compared with placebo in relieving headache. This reduced efficacy of sumatriptan in the second and third attacks may be due to a high incidence of vomiting induced by the high dose of dispersible formulation and also by the bitter taste of the tablets. In addition, there was an increase in placebo response in attacks 2 and 3 compared to the first attack.
Acute Disease↗
Effects of a novel prostaglandin, 8-epi-PGF2 alpha, in rabbit lung in situ.
We determined the effects of 8-epiprostaglandin (PG) F2 alpha, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, on pulmonary vascular tone, its potency, and its mechanism of action. 8-Epi-PGF2 alpha (0.5-20 micrograms) was injected into the pulmonary artery (PA) catheter of 10 rabbits whose lungs were perfused in situ with Krebs-Henseleit buffer solution with 3% bovine serum albumin. PA pressure increased from a baseline of 13.5 +/- 0.6 to 25.6 +/- 2.0 cmH2O with 20 micrograms 8-epi-PGF2 alpha. 8-Epi-PGF2 alpha caused a rapid rise in PA pressure followed by a gradual decline over 40-60 min to baseline levels. Double vascular occlusion revealed a twofold increase in arterial resistance at peak rise in PA pressure. The rise in PA pressure with 20 micrograms 8-epi-PGF2 alpha was fivefold greater than with 20 micrograms of the cyclooxygenase-derived prostaglandin PGF2 alpha. The PA pressure response to 8-epi-PGF2 alpha was not altered by either cyclooxygenase block-ade with 150 microM meclofenamate or alpha-receptor blockade with 70 microM phentolamine, but was fully prevented by 40 microM SQ 29548, a thromboxane receptor antagonist. We conclude that in rabbits 8-epi-PGF2 alpha is a potent vasoconstrictor of the pulmonary vasculature, which appears to be due to the activation of SQ 29548-responsive thromboxane receptors.
Animals↗
Changes in the nucleic acids and protein content of the ovaries in relation to a short and long photoperiod in a freshwater catfish Heteropneustes fossilis (BL).
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Animals↗