Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X.

Human blood-coagulation factor X (hBCFX) is a serine protease zymogen which participates in the middle phase of blood coagulation. Recently, we and others have reported the cDNA sequences. At present, partial hBCFX gene structure is available. In this paper, we report the isolation of two genomic clones, the X-emb lambda phage clone encoding exons 1 and 2 of the hBCFX gene, and the X-cos cosmid clone encoding exons 2-8. The restriction map of the hBCFX region spans 55 kb. The gene itself was found to be 27 kb long. The sequence of the 5' region of the hBCFX gene has been determined and reveals an ATTTG pentanucleotide, which also occurs in a similar location in the genes encoding factors VII, IX, protein CC and C prothrombin, suggesting that this motif might be of importance in the regulation of these genes.

A simple and easy-to-assemble device for polymerase chain reaction.

In this paper we describe an efficient polymerase chain reaction device which is easy to assemble and requires minimal investment in dedicated equipment. The polymerase chain reaction device consists of three waterbaths, three dual-head peristaltic pumps, an electronic timer and a fabricated water jacket capable of holding microcentrifuge tubes. This device has been successfully used to amplify human factor X genomic DNA in our laboratory.

Dissociation of calcium and sodium clearances in patients with hypoparathyroidism by infusion of chlorothiazide.

Previous reports have identified a deficient hypocalciuric response to chronic treatment with thiazide diuretics in patients with hypoparathyroidism. The present study was designed to ascertain if the acute response to thiazide diuretics is impaired in hypoparathyroidism. Five normal subjects and five patients with hypoparathyroidism were studied with the renal clearance technique during water diuresis. In normal subjects the clearance of calcium/clearance of sodium was 0.98 +/- 0.14 before, and 0.33 +/-0.03 during the intravenous infusion of chlorothiazide. In patients with hypoparathyroidism the corresponding ratios were 1.69 +/- 0.27 and 0.57 +/- 0.10. In both groups the drug-induced fall in clearance was 65% of control. The concentration of chlorothiazide in plasma and its rate of excretion were comparable in both groups. It is concluded that the acute action of thiazides is not impaired in hypoparathyroidism.

Protein degradation and related enzyme profiles on a sublethal toxicity of aldrin in the tissues of Rana hexadactyla.

The effects of a sublethal toxicity of aldrin were studied in the brain, muscle, kidney, and intestine of Rana hexadactyla after 1, 2, and 4 weeks of exposures. Aldrin toxicity resulted in a depletion of protein, an increase in amino acid levels, and an enhancement of the activity of protease and amino transferases. The changes were maximum after a 4-week exposure.

Toxicity of the pyrethroid insecticide fenvalerate to a fresh water fish, Tilapia mossambica (Peters): changes in glycogen metabolism of muscle.

Toxic effects of a pyrethroid insecticide, fenvalerate, on fish muscle glycogen metabolism were investigated. Estimations were made after 10 and 20 days of exposure, and altered muscle glycogen metabolism was observed. The changes included a significant (P less than 0.001) decrease in the levels of glycogen, pyruvate, maleate dehydrogenase (MDH), succinate dehydrogenase (SDH), and phosphorylase a, b, and ab activities, while elevated levels of lactic acid, aldolase, and lactate dehydrogenase (LDH) activity were observed under fenvalerate intoxication. There was a decrease in opercular movement and oxygen consumption with an increase in concentration of fenvalerate.

Aldrin toxicity on amphibian neuronal, hepatic and muscular tissue oxidative enzymes.

Aldrin toxicity was studied in the brain, liver and muscle of Rana hexadactyla. It was observed that sublethal concentration of aldrin inhibited the activities of SDH, MDH and ICDH while it elevated the activities of LDH and G-6-PDH. The increase and the decrease was progressive with the exposure period. The changes were attributed to the induced toxic effects such as oxygen distress, energy crisis and microstructural changes.

Dietary vitamin-E modulates antioxidant defence system in giant freshwater prawn, Macrobrachium rosenbergii.

The objectives of the present study were to determine the effect of supplementary vitamin-E (200, 400 and 600 mg/kg feed) on lipid peroxidation (LPX) and antioxidant defence system in gills and hepatopancreas of the freshwater prawn, Macrobrachium rosenbergii. Results indicated that vitamin-E inhibited LPX in the hepatopancreas in a comparatively lower dose than gills. Superoxide dismutase (SOD) activity was decreased significantly in gills in response to all the three supplemented diet, but in hepatopancreas decrease was observed only in response to higher doses of vitamin-E (400 and 600 mg/kg feed). Catalase (CAT) activity was reduced significantly only in gills but not in hepatopancreas. While glutathione peroxidase (GPX) activity was significantly elevated in the hepatopancreas by vitamin-E, its activity remains unaltered in gills. On the contrary, glutathione reductase (GR) activity was decreased in gills but that of hepatopancreas was constant. Glutathione (GSH) content of both gills and hepatopancreas was substantially elevated in the vitamin-E supplemented prawns. Although the ascorbic acid (ASA) content of gills was unchanged by vitamin-E, its level elevated significantly in hepatopancreas. Thus the findings of the present investigation suggest that dietary vitamin-E is capable of reducing LPX level and can modulate antioxidant defence system in gills and hepatopancreas, nevertheless, the response is highly tissue specific. It is further observed that highest dose of vitamin-E (600 mg/kg feed) could not render much additional protection in both the tissues.

Thyrotoxicosis in pregnancy:: A case report.

There are various causes of hyperthyroidism in pregnancy such as Graves' disease and gestational thyrotoxicosis. The thyroid stimulation results from the excessive levels of circulating human chorionic gonadotropin (hCG) produced by the trophoblastic tissue in both hydatidiform moles and choriocarcinoma. We present a pregnant patient with hydatidiform mole who presented with hyperthyroidism that resolved after evacuation of the mole.

Use of a modified angioplasty balloon catheter in the dilatation of tight biliary strictures.

Nineteen biliary strictures were dilated using a modified angioplasty balloon catheter to allow insertion of a 10F prosthesis. In each instance biliary strictures were successfully dilated which had previously been too tight to widen with standard endoscopic biliary dilating catheters. Eleven patients had malignant hilar strictures, four malignant distal common bile duct strictures, and four benign strictures. There were no complications as a result of the procedure and satisfactory biliary drainage was established in all patients. We conclude that tight biliary strictures can be successfully dilated using a modified angioplasty balloon catheter.

Human factor IXLincoln Park: a molecular characterization.

Using genomic DNA prepared from peripheral blood samples of a patient with factor IX deficiency, all eight exons as well as sequences around the splice junctions and putative promoter region of human factor IX DNA have been subjected to polymerase chain reaction (PCR) amplification and sequenced. Comparison of these sequences with normal factor IX gene sequences revealed an insertion in exon VIII that resulted in the alteration of 11 amino acids and the addition of 23 amino acids, all at the carboxy terminal of factor IX. This insertion destroys an Msp I restriction site; carrier detection and antenatal diagnosis in affected kindreds can be performed by testing for the absence of this site. This is the first characterization of a mutation in which insertion in the carboxy terminal region of factor IX causes factor IX deficiency. The genetic change in factor IX in this patient is called Factor IXLincoln Park.

Endoscopic biliary stenting in a district general hospital.

During a 48 month period to December 1990, 367 patients, median age 75 years, with obstructive jaundice caused by common bile duct stones (201), malignant biliary obstruction (148), and benign biliary strictures (18), underwent therapeutic endoscopic retrograde cholangiopancreatography. Endoscopic biliary stenting and drainage was achieved in 343 of 367 patients attempted (93%), seven patients requiring a combined percutaneous endoscopic approach. Endoscopic stenting failed in 24 patients because of malignant duodenal infiltration (10), Billroth 2 gastrectomy (6), tight and extensive biliary strictures (6), peripapillary diverticulum (1), and technical failure (1). Prolonged follow up was available in 91% (311 of 343). The 30 day mortality was 5% (17 of 343), which included two procedure related deaths (0.6%) from fulminant pancreatitis and major sphincterotomy site bleeding. Early complications occurred in 14% (48 of 343) and late complications occurred in 11.9% (35 of 294) patients, as of the original 343, 17 had died within 30 days and another 32 were lost to follow up. Eighty patients with incomplete bile duct clearance and eight patients with benign biliary strictures had biliary stents inserted for 12-48 months (median 30). Endoscopic biliary stenting services are necessary in a district general hospital with technical success, death and morbidity rates comparable to other studies.

Polychromatic analysis: new applications of an old technique.

As a means of correcting for many potential interferences, polychromatic analysis offers an effective alternative to either sample pretreatment or separate blank determinations. Because of the differing spectral characteristics of each interfering species, and the nature of these interferences (static vs. kinetic), the application of polychromatic analysis must be optimized for each analytical procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedures (also based on polychromatic analysis). By using a second wavelength measurement, the analyst can also verify the accuracy of an analytical measurement in selected situations. These applications of polychromatic analysis are a departure from conventional analytical procedures in allowing for correction of various interferences in the actual reaction mixture, without making assumptions about the recovery of analyte during pretreatment or the equivalent absorptivity of interferent in both blank and assay solutions.

Current status of the anticoagulant hirudin: its biotechnological production and clinical practice.

Hirudin is a potent thrombin inhibitor originally derived from the medicinal leech, Hirudo medicinalis. Owing to its high affinity and specificity for thrombin, hirudin has been intensively investigated for research and therapeutic purposes. The investigation of hirudin has contributed greatly to the understanding of the mode of action of thrombin and the clotting system. Hirudin and several hirudin analogues have also been demonstrated to have several advantages as a highly specific anticoagulant over the most widely used drug, heparin. Due to the great demand for hirudin in physicochemical and clinical studies, various recombinant systems have been developed, using bacteria, yeasts, and higher eukaryotes, to obtain the biologically active hirudin in significant quantities. After 10 years of clinical applications, two recombinant hirudins and a hirudin analogue have gained marketing approval from the United States Food and Drug Administration, for several applications. Clinical trials are currently ongoing for other treatments for thrombotic disease. As a consequence, it is conceivable that hirudin may expand its therapeutic utility over heparin in the near future.

Molecular characterization of human factor XSan Antonio.

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.