Kinetic aspects of release of fibrinopeptides AP and AY by human alpha-thrombin

The time-dependent release by human alpha-thrombin of the physiological variants of fibrinopeptide A, i.e. fibrinopeptide A-3-phosphate (FPAP) and des-Ala-fibrinopeptide A (FPAY), has been measured in order to study the kinetic pathway for their hydrolysis. The best-fit kinetic model for the release of FPAP and FPAY is consistent with a simple pseudo first-order reaction, as observed with FPA. These findings indicate that FPAP and FPAY are also released from intact fibrinogen before release of FPB. The values of the specificity constants, i.e. k(at)/Km, for the enzymatic reaction between thrombin and the various alpha-chains showed that AP alpha- and AY alpha-chain are hydrolyzed with a 0.2-and 0.4-fold higher specificity constant respectively than that of A alpha-chain. Such minor differences observed with the various chains suggest that the 1-3 NH2-terminal residues of the chain do not significantly contribute to the catalytic efficiency of thrombin toward the alpha-chains of fibrinogen.     

Stability of parenteral midazolam in an oral formulation.

Midazolam is increasingly being used for oral sedation in pediatric dentistry. Unfortunately, it is available only as a parenteral formulation in Canada and the United States. Preparation of the parenteral solution for oral use is not uniform and leads the clinician to question the stability of this drug when used in conjunction with these vehicles. Therefore, the purpose of this study was to investigate the chemical stability of parenteral midazolam as an oral formulation to determine its expiry date. This was evaluated using a validated stability-indicating liquid chromatographic method. Midazolam was diluted in orange-flavored syrup to yield concentrations of 0.35, 0.64, and 1.03 mg/ml and then stored at room temperature. Samples were drawn on each of 9 study days (0, 1, 2, 6, 7, 9, 13, 21, and 102) and chromatographed. On each study day, solutions were inspected visually for changes in color, clarity, and appearance of particulate matter. Midazolam concentrations were considered within acceptable limits if they were not less than 90% of the initial concentration. Over the 102-day study period, there was no significant change in concentration in any of the solutions. On day 102, the remaining midazolam was within 7% of the day zero concentration. Therefore, these formulations of midazolam are stable at room temperature for a period of 102 days and would be suitable for clinical use.     

Effect of mast cell chymase on the morphology of thyroid cells in vitro.

We studied the effect of mast cell chymase on the thyroid cells in culture. Rat serosal mast cells, similar functionally to connective tissue mast cells, were obtained after lavage of the peritoneal cavity and lyzed by freezing. The resulting lysate was used as crude enzyme preparation. Mast cell chymase was purified from the crude preparation by anion exchange chromatography. Crude and purified chymase incubated with thyroid cells induced cellular retraction, the appearance of long processes and gradual cell detachment from the substratum. The effect of the enzyme was not cytotoxic. The immunofluorescence studies of thyroid cells showed a decreased amount of polymerized actin and tubulin after incubation with chymase. Neutral protease inhibitor abolished the effect of crude and purified chymase on thyroid cell morphology. The above findings suggest that mast cell chymase may have a function in the control of cell morphology and cell-matrix interaction.     

Correlations between follicular fluid steroid analysis and maturity and cytogenetic analysis of human oocytes that remained unfertilized after in vitro fertilization.

OBJECTIVE: Is there any correlation between follicular fluid (FF) steroid levels and the occurrence of cytogenetic abnormalities in unfertilized human oocytes? DESIGN: Cytogenetic analysis was carried out on 397 oocytes, and the steroid content of 104 corresponding FF was analyzed using high-pressure liquid chromatography. Ovarian stimulation was performed by clomiphene citrate and human menopausal gonadotropin (hMG) or by hMG combined with a gonadotropin-releasing hormone agonist (GnRH-a) pretreatment. RESULTS: Oocyte maturity was correlated with an increasing FF progestin content and a significant decrease of androstenedione (A) levels. Chromosomal analysis revealed 84 of all oocytes to be abnormal (polyploid or aneuploid and/or prematurely condensed chromosomes present). In this group, A levels and A to estradiol ratios were significantly higher. Although progestin levels were higher in GnRH-a/hMG cycles, the incidence of oocyte normality was not different between the two stimulation schemes. More abnormal oocytes were found in patients with good sperm morphology. CONCLUSIONS: Oocyte abnormality correlates with higher A levels in the corresponding FF. Oocyte fertilization is also determined by intrinsic oocytic factors other than maturity.     

Sonography detection of small intra-abdominal fat variations.

The aim of this study was to check the reliability of sonography in measuring small variations in quantities of subcutaneous and intra-abdominal fat. Twenty-six obese women (BMI 39 +/- 6) underwent a 15 day very low calorie diet. The study included, both before and after very low calorie diet, computed tomography measurements of total (AT), visceral (VAT) and subcutaneous (SAT) adipose tissue areas, visceral/subcutaneous area ratio (V/S), waist/hip circumference ratio measurements (W/H), and ultrasound measurements of abdominal subcutaneous skin-muscle thickness, intra-abdominal muscle-aorta thickness and intra-abdominal/subcutaneous thickness ratio. Weight reduction was from 101 +/- 17 to 95 +/- 16 kg (P less than 0.001). W/H dropped from 0.83 +/- 0.06 to 0.82 +/- 0.07 (n.s.). VAT dropped from 158 +/- 72 to 134 +/- 61 cm2 (P less than 0.005), SAT from 572 +/- 151 to 566 +/- 164 cm2 (n.s.) and V/S from 0.29 +/- 0.15 to 0.25 +/- 0.11 (P less than 0.01). Abdominal subcutaneous fat thickness decreased from 36 +/- 8 to 35 +/- 10 mm (n.s.), intra-abdominal thickness from 39 +/- 25 to 20 +/- 20 mm (P less than 0.001) and intra-abdominal/subcutaneous from 1.1 +/- 0.7 to 0.8 +/- 0.6 (P less than 0.005). VAT measurement accurately identified small intra-abdominal fat variations. W/H could not evaluate visceral fat loss, because of simultaneous decreases in waist and hip circumferences. Ultrasound was able to measure small reductions in intra-abdominal fat.     

Deep vein thrombosis in surgery]

Deep vein thrombosis is still an underestimated and often unrecognised diseases both in Italy and abroad despite being far from rare and in spite of its severe complications and often disabilitating sequelae. It is a disease which is becoming more frequent due to increased life expectation, the larger number of operations on elderly patients, and the increased frequency of limb injuries. General and specialised surgery is often hampered by this complication. This has raised interest in a more detailed knowledge of the mechanisms leading to DVT, and the correct employment of the latest equipment which enable diagnosis to be made and daily monitoring of the disease during its evolution. But the greatest efforts must be reserved for preventive measures in order to achieve a statistically significant reduction in the incidence of the disease in operated patients. It is to be hoped that a greater awareness of thromboembolic diseases will allow this to be achieved in the future.     

Measurement of short-term 11C-thymidine activity in human head and neck tumours using positron emission tomography (PET).

Tumour uptake of 11C-thymidine labeled in the methyl position was assessed after intravenous injection in 13 patients with head and neck tumours. This activity was compared to other tracers such as C15O, 13NH3 and C15O2. In every single case a 'positive' tumour image after injection of 11C-thymidine was obtained. Time-activity curves showed the initial activity to be followed by a rapid decrease over the first 10-15 min with an apparent plateau thereafter. A similar level of uptake was found in normal salivary gland regions and myocardium, while higher activities were noted in liver and kidney parenchyma. It is suggested that both blood flow and cellular metabolism can influence 11C-thymidine imaging in this class of human tumours.     

Hb-M "Hyde Park": a de novo mutation, identified by mass spectrometry and DNA analysis.

BACKGROUND. Structural hemoglobinopathies usually are inherited as autosomic dominant traits; de novo mutations are uncommon. Analytical and preparative procedures for the characterization of an abnormal hemoglobin are complex and time-consuming. Mass spectrometer analysis allows a rapid identification of the amino acid substitution. METHODS AND RESULTS. A cyanotic 7-year-old girl was found to have 16% methemoglobin. Laboratory data showed the presence of an abnormal hemoglobin, which was isolated by collecting the abnormal peak from DEAE and globin chains from CM52. The amino acid substitution was rapidly identified by FAB mass spectroscopic analysis, leading to the recognition of HbM Hyde Park. These data were confirmed by molecular analysis (Southern blot and DNA sequencing). Neither the parents nor a sister showed any abnormality; non-paternity was excluded by blood group serology and HLA typing. CONCLUSIONS. This is a case of HbM Hyde-Park arising as a de novo mutation. FAB mass spectroscopic analysis is a rapid and useful analytical method for identifying aminoacid substitution.     

Reactivated recombinant plasminogen activator inhibitor-1 (rPAI-1) effectively prevents thrombolysis in vivo.

The effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (approximately 2 units of t-PA neutralizing activity per micrograms protein) and reactivated rPAI-1 (approximately 150 units/micrograms). Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity approximately 0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rehabilitation after amputation for vascular disease: a follow-up study.

Rehabilitation of one hundred and twenty eight patients with lower limb amputation performed for vascular disease from 1979 to 1987 was assessed. Arteriosclerotic occlusive disease was the most frequent cause of amputation (85.9%). Sixty seven patients (52.3%) were diabetic. Early and late results were analysed. For long-term follow-up evaluation, Univariate method of Kaplan-Meyer product limit was employed. Multifactorial analysis was used to assess factors influencing mortality. On immediate evaluation of rehabilitation with a prosthesis 85.2% of patients were successfully fitted. On long term evaluation 47.8% of below-knee and 22.1% of above-knee amputees were alive and using the prosthesis full time at five years of follow-up (p = 0.0026). Opposite limb preservation at five years was 69.5% for diabetics and 90.2% for non-diabetics, respectively (p = 0.0013). Survival rate at five years was 42.4% for diabetics, and 85.0% for non-diabetics (p = 0.0002). On multifactorial analysis diabetic patients showed a risk of late mortality six times greater than non-diabetics. In conclusion rehabilitation after vascular amputation is feasible in a large number of patients, despite a limited life span. Diabetes represents a major risk factor both for life and for the opposite limb. Knee preservation is an important factor for better rehabilitation.