Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.
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PMID:Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin. 886 82

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.
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PMID:Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78. 905 3

We investigated the lowest effective dosage of low molecular weight (LMW) heparin for hemodialysis in comparison to unfractionated (UF) heparin. Initial hemodialysis sessions were undertaken in 10 uremic patients with UF heparin of the dose habitually required for each patient. Four-hour hemodialysis sessions were then undertaken with LMW heparin (nadroparin) in a single bolus (200 anti-Xa unit Institut Choay/kg [aXaU IC/kg], 175 aXaU IC/kg, 150 aXaU IC/kg, or 125 aXaU IC/kg; two sessions for each dosage). Anti-Xa levels and activated partial thromboplastin time (APTT) were monitored hourly during dialysis. Fiber bundle volume of dialyzer was measured before and after dialysis. Urea clearance was determined at the onset and completion of dialysis. There were no episodes of excessive bleeding, clotting of dialyzers, or clots in air traps with UF heparin or LMW heparin. A 35% increase in APTT above baseline was observed in all dialysis sessions 1 hour after LMW heparin bolus, but the APTT decreased rapidly thereafter. The anti-Xa levels exceeded 0.5 U/mL for all sessions using LMW heparin irrespective of the dosage. No significant reduction of urea clearance was found in dialysis with either UF or LMW heparin. No reduction of fiber bundle volume of dialyzer was observed in dialysis with either UF or LMW heparin, although a small reduction (3%) was observed in dialysis with LMW heparin at 125 aXaU IC/kg. We concluded that the use of LMW heparin for hemodialysis is safe and effective as compared with UF heparin. The lowest effective dosage can be reduced to 125 aXaU IC/kg in high-risk patients to reduce hemorrhagic complications.
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PMID:Use of low-dose low molecular weight heparin in hemodialysis. 915 10

1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5'-(biotinamido)pentylamine into casein and prevented gamma-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, L-gamma-glutamyl-4-nitroanilide.
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PMID:Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii. 921 Apr 3

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.
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PMID:An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli. 930 71

We hypothesized that the success of postoperative blood conservation after acute normovolaemic haemodilution (NVHD) is influenced by the extent of intraoperative bleeding and surgical trauma, and the timing of autologous blood transfusion. As total knee replacement is associated with minimal intraoperative but extensive postoperative blood loss, this procedure is ideally suited to acute NVHD. Therefore, to test our hypothesis, 30 patients undergoing elective total knee replacement were enrolled in a prospective, randomized, controlled study. In groups NVHD-2 and NVHD-6, before induction of anaesthesia patients were bled to a target packed cell volume (PCV) of 28-30%, and in the post-anaesthesia care unit autologous blood was transfused over a 2-h period terminating after operation at 2 and 6 h, respectively. In the control group, NVHD was not performed. After operation, platelets, fibrinogen, prothrombin and partial thromboplastin time, and liver function, urea and electrolytes were measured and compared with preoperative baseline values. Significantly (P < 0.024) more allogeneic blood was transfused in the control group (21 u.) compared with either group NVHD-2 (7 u.) or group NVHD-6 (5 u.). In the control group, despite the allogeneic blood transfusion, postoperative PCV decreased until day 4 after operation. Coagulation profile, liver function and urea and electrolytes concentrations were unaffected by the method of treatment. We conclude that for total knee replacement, acute NVHD is an effective blood conservation strategy. However, there was no difference in allogeneic blood administration between the two NVHD groups. Coagulation and liver function, and urea and electrolyte concentrations were unaffected by treatment.
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PMID:Acute normovolaemic haemodilution decreases postoperative allogeneic blood transfusion after total knee replacement. 938 48

The study enclosed 30 dogs with severe or end stage chronic renal failure showing distinctly increased concentrations of urea and creatinine. In most of the 15 cases, where a pathologic-histological investigation of the kidney was carried out, a glomerulonephritis was observed (n = 11), partly accompanied by an interstitial nephritis or tubulonephrosis, respectively. Compared to the control group (n > or = 100) the most significant changes were the distinctly increased concentrations of fibrinogen (6.22 [2.95-11.83] g/l; median [minimum-maximum]) and activity of the coagulation factors V (median = 165%), VII (198%), X (176%), VIII:C (154%), and IX (178%) as well as of protein C (147%) (each: p < 0,0001 [Mann-Whitney-Test]). Thereby, the latter does not contribute to hypercoagulability in dogs with chronic renal insufficiency. The activity of antithrombin III was clearly diminished (69[41-112]%), and was closely correlated to the albumin concentration (r = 0.7000; p = 0.001) reflecting the joint renal loss of these proteins of nearly a size. Surprisingly a reagent dependent prolongation of the activated partial thromboplastin time appeared. Against that, a corresponding diminution of the activity of single coagulation factors was demonstrated only seldom, reaching only a small degree, and related almost exclusively to the contact activating system. Compared to the respecting reference range the concentrations of soluble fibrin or fibrin degradation products were increased in 15 or 21 (of 29) samples, and were, thereby, significantly higher than in the control group (p < 0.0001). This reflects the enhanced intravascular coagulation occurring possibly limited to the region of the renal alteration that should be more noticed in therapy.
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PMID:[Changes in hemostasis in dogs with chronic renal insufficiency]. 945 45

OBJECTIVES. To review the available evidence on the value of routine preoperative testing in healthy or asymptomatic adults. To assess the completeness of existing reviews of preoperative testing and how applicable their conclusions are to the UK. To identify areas for further research. HOW THE RESEARCH WAS CONDUCTED. The databases Medline, Embase, Biological Abstracts, Science Citation Index and HealthSTAR were thoroughly searched for relevant articles which were then classified and appraised. The databases of the Centre for Reviews and Dissemination (DARE and NHS Economic Evaluations Database) and the Cochrane Collaboration (the Cochrane Library) were also used to verify the completeness of the search. In this review, 'routine' tests are defined as those ordered for an asymptomatic, apparently healthy individual in the absence of any specific clinical indication, to identify conditions undetected by clinical history and examination. RESEARCH FINDINGS. No controlled trials of the value of the following routine preoperative tests have been published. All available evidence reports the results of case-series. CHEST X-RAY. Few studies allow the outcome of routine chest X-rays to be distinguished from those of indicated chest X-rays, and fewer have gone beyond abnormality yields to examine the impact on clinical management. Findings from routine preoperative chest X-ray are reported as abnormal in 2.5-37.0% of cases, and lead to a change in clinical management in 0-2.1% of patients. The effect on patient outcomes is unknown. Both abnormality yield and impact on patient management rise with age and poorer American Society of Anesthesiologists (ASA) status. The limited evidence on the value of a chest X-ray as a baseline measure suggests that it will be of value in less than 9% of patients. ELECTROCARDIOGRAPHY. The findings from routine preoperative electrocardiograms (ECGs) are abnormal in 4.6-31.7% of cases, and lead to a change of management in 0-2.2% of patients. The effect on patient outcomes is unknown. The proportion of abnormal tests rises with age and worsening ASA status. The predictive power of preoperative ECGs for postoperative cardiac complications in non-cardiopulmonary surgery is weak. There is no evidence to support the value of recording a preoperative ECG as a 'baseline.' HAEMOGLOBIN MEASUREMENT AND BLOOD COUNTS. Routine preoperative measurement shows that the haemoglobin level may be lower than 10-10.5 g/dl in up to 5% of patients, but that it is rarely lower than 9 g/dl. The routine test leads to a change of management in 0.1% to 2.7% of patients. Routine preoperative measurement shows that the platelet count is abnormally low in less than 1.1% of patients, and that platelet count results rarely if ever lead to change in management of patients. Routine preoperative white blood cell count is abnormal in less than 1% of patients, and rarely if ever leads to change in management of patients. TESTS OF HAEMOSTASIS. Abnormalities of bleeding time, prothrombin time and partial thromboplastin time are found in up to 3.8%, 4.8% and 15.6% of routine preoperative tests, respectively. The results of these tests very rarely lead to change in the clinical management of patients. BIOCHEMISTRY. In routine preoperative tests of serum biochemistry, abnormal levels of sodium or potassium are found in up to 1.4% of patients, and abnormal levels of urea or creatinine are found in up to 2.5% of patients. Abnormal levels of glucose are found in up to 5.2% of patients. These abnormalities rarely lead to change in clinical management of patients. URINE TESTING. Routine preoperative urinalysis finds abnormal results in 1-34.1% of patients, and leads to a change of management in 0.1-2.8% of patients. The only abnormality that leads to a change in management of patients is the finding of white blood cells in the urine. There is no good evidence that preoperative abnormal urinalysis is associated with any postoperative complication in non-urinary tract surgery. (ABSTRACT TRUNCATED)
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PMID:Routine preoperative testing: a systematic review of the evidence. 948 55

Menorrhagia--technically defined as menses lasting longer than 7 days or a blood loss volume in excess of 60 to 80 mL--is one of the most common gynecologic complaints. It has been reported that 15% to 20% of otherwise healthy women experience debilitating menorrhagia. In the past, definitive treatment for abnormal uterine bleeding has been either abdominal or vaginal hysterectomy. Alternatives to hysterectomy are now stressed in light of the fact that nearly 50% of uterine specimens obtained during hysterectomies for menorrhagia are disease-free on pathologic examination. Etiologies are generally either endocrinologic or organic. Among the organic causes are coagulation disorders; organ dysfunction, such as liver or renal disease; endometrial hyperplasia; infection; iatrogenic causes, such as chemotherapy, anticoagulants, steroid therapy, and use of IUD; and anatomic causes, which include uterine leiomyoma, endometrial polyps, and pregnancy. Besides the history and physical exam, useful lab tests include CBC, serum pregnancy test, cervical specimens for gonorrhea and chlamydia, Pap smear, thyroid function tests, serum transaminases, luteinizing hormone, follicle-stimulating hormone, estradiol, prothrombin time, activated partial thromboplastin time, bleeding time, serum prolactin, quantitative beta human chorionic gonadotropin, blood urea nitrogen, serum creatinine, and adrenal function tests. Since many lesions are missed in office sampling or dilation and curettage, imaging studies, including ultrasound and hysteroscopy, can be useful in diagnosing the cause of menorrhagia. Medical treatments include drugs such as NSAIDS, progestins, oral contraceptives, and gonadotropin-releasing hormone agonists. Surgical modalities range from the relatively simple, such as D & C, to major surgical procedures such as hysterectomy. Laser ablation and thermal balloon ablation are promising new procedures.
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PMID:Contemporary Concepts in Managing Menorrhagia. 974 66

The objective of this study was to determine whether a thrombin inhibitor (PPACK) and a factor Xa inhibitor (GGACK) either alone or in combination can anticoagulate whole blood without biasing the analysis of several critical care analytes. Whole blood clot time was used to assess anticoagulant efficacy. The analytical biases mediated by the anticoagulants on glucose, urea, creatinine, electrolytes, amylase, lactate dehydrogenase, creatine kinase, ionized calcium and pH were assessed. The protease inhibitor mixture (100 micrommol/l PPACK + 500 micromol/l GGACK) was more a potent anticoagulant than the individual agents at the same concentrations. Both PPACK and GGACK, alone and in combination, reduced the activity of creatine kinase and amylase by 3-10% while the remaining critical care analytes were less affected. In conclusion, PPACK and GGACK mixtures can effectively anticoagulate whole blood, but the mixtures exert pre-analytical influences that limit the analytical versatility of these novel plasma-matrices.
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PMID:Evaluation of the thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK) with the factor Xa inhibitor 1,5-dansyl-L-glutamyl-L-glycyl-L-arginine chloromethylketone (GGACK) as anticoagulants for critical care clinical chemistry specimens. 1009 May 27


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