Synthesis of coagulation factor V by cultured aortic endothelium

Bovine aortic endothelium has been examined with respect to the synthesis of coagulation factor V. After cultured cells reached confluency, samples of supernatant culture media and solubilized cells were analyzed for factor V in a two-stage bioassay and in a double- antibody radioimmunoassay. In addition, preconfluent cells were pulsed for 4 days with 35S-methionine in methionine-free media. After the 4- day pulse, supernatant media were chromatographed on a factor V monoclonal antibody-Sepharose resin to isolate 35S-labeled factor V. The isolated material and 125I-factor V standards were analyzed by electrophoresis and autoradiography. The bioassay indicated an increase, with time, of unactivated factor V in the culture supernatant, whereas solubilized cells were negative for factor V. The radioimmunoassay indicated an increase, with time, of factor V antigen in the culture supernatants, and the solubilized cells yielded a constant level of antigen per cell. Autoradiograms of electrophoretograms of immunoadsorbed 35S-culture supernatant with 125I- factor V/Va standards revealed labeled proteins with electrophoretic mobilities compatible with 125I-factor V/Va standards. The data obtained from three different sources-bioassay, radioimmunoassay, and 35S-methionine incorporation-all indicate that factor V is synthesized by cultured bovine aortic endothelium.     

CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.     

Prevalence of widespread pain and associations with work status: a population study

Background  

This population study based on a representative sample from a Swedish county investigates the prevalence, duration, and determinants of widespread pain (WSP) in the population using two constructs and estimates how WSP affects work status. In addition, this study investigates the prevalence of widespread pain and its relationship to pain intensity, gender, age, income, work status, citizenship, civil status, urban residence, and health care seeking.     

Trends in practice characteristics: analyses of 19 periodic surveys (1987-1992) of Fellows of the American Academy of Pediatrics

OBJECTIVE: To examine 6 years of practice characteristics data of Fellows of the American Academy of Pediatrics (AAP), focusing on sex differences for specialty area, primary activity, practice setting, and practice location. METHODS: We analyzed data from 19 Periodic Surveys that were fielded between 1987 and 1992. The Periodic Survey is used to survey AAP members regularly about current issues in pediatric practice. There are no duplicate respondents in these analyses of the first 19 Periodic Surveys. We collapsed the 19 surveys into the years in which they were fielded, and analyzed sex differences for each of the 6 years. In addition, we ran logistic regressions on several questions, including all 16 868 respondents, to examine how the characteristics of the specialty have been affected by the increase in the number of female pediatricians, controlling for survey year, age of respondents, and specialty area practiced. RESULTS: The proportion of nonresident AAP members who are female has grown throughout the 6 years; in 1987, 26.9% were female, and in 1992, 36.4% were female. For 5 of the 6 years there were sex differences in specialty area, usually concerning pediatric subspecialties. Substantial sex differences occurred in primary activity, in which each year women were more likely than men to be salaried. Men were more often in group practices, whereas women were generally more likely to practice in hospitals or clinics. Logistic regression demonstrated that there are sex differences in practice characteristics across time, but there is also a substantial change in practice characteristics accountable to survey year, eg, a pediatrician of either sex was 75% more likely to be salaried in 1992 than in 1987. CONCLUSIONS: Throughout the 6-year period, AAP members became increasingly more likely to practice general pediatrics, to be salaried, and to be younger-all effects independent of sex, all effects stronger for females. Rapid transformations in the health care system will likely reduce current sex differences in practice characteristics of the future.     

Onychomycosis in Icelandic children

BACKGROUND: Onychomycosis is a rare disorder in children. Few studies exist on the incidence or prevalence of onychomycosis in children. OBJECTIVE: To examine the epidemiology of childhood onychomycosis in Iceland during the period 1982-2000. METHODS: Results from all mycological samples taken from children in Iceland from 1982 to 2000 were examined. Information about the requesting physician, unique social security number, date of birth, sex, results of culture and microscopy were registered. Growth of a dermatophyte was taken as an indication of a case of onychomycosis. RESULTS: During the period 1982-2000 a total of 493 samples from 408 Icelandic children, aged 0-17 years, were examined. Dermatophytes were cultured from 148 (30.0%) samples. During the period 1982-85, the mean annual incidence of positive cultures was 1.65 per 100,000, increasing to 21.30 per 100,000 for the years 1996-2000. Trichophyton rubrum was the dominating organism and was found in 102/148 cases positive for the growth of a dermatophyte. The incidence of positive dermatophyte cultures increased with age and was found in eight children aged 0-4, and in 57 children aged 10-14 years. CONCLUSION: Onychomycosis is rare in children, but increases with age. It seems that onychomycosis increased during the study period, but it is not clear if this was due to a true increase in the prevalence of onychomycosis or an increased awareness of onychomycosis, or both.     

Effects of S-carboxymethyl-l-cysteine on pulmonary sialyl transferase activity in vitro, in healthy and in sulphur-dioxide-induced bronchitic rats

S-carboxymethyl-L-cysteine (carbocysteine) improves the visco-elastic properties of bronchial mucus in vivo, possibly as a result of an increase in the relative proportions of sialomucins in bronchial mucus. Carbocysteine was therefore studied in vitro and ex vivo in both normal and bronchitic rats on pulmonary sialyl transferase, responsible for the addition of sialic acid to mucus glycoproteins. Bronchitis was induced in male Sprague-Dawley rats by repeated exposure to sulphur dioxide for two weeks. During this time they received either 500 mg kg-1 day-1 carbocysteine or its vehicle by the oral route. Rats not being exposed to SO2 received the same treatment. The animals were then killed, and subcellular fractions prepared by differential centrifugation of lung homogenates. Sialyl transferase was assayed using CMP-14C sialic acid as substrate and desialysed fetuin as exogenous acceptor. Enzyme activity was located in both the (Golgi-containing) 10,000 g and 100,000 g pellets with minor activity in the cytosolic supernatants. When tested in vitro between 10(-6) and 10(-3) M, carbocysteine had no effect on sialyl transferase activity in microsomes taken from healthy or bronchitis rats. Repeated administration of carbocysteine was without effect on the sialyl transferase activity in 10,000 g pellets taken from healthy rats. However, in bronchitic rats there was a small but statistically significant (P less than 0.05) increase in enzymic activity in the treated group compared to the animals receiving the vehicle. There was no difference in the activity of the microsomal enzyme compared to vehicle-treated controls in either healthy or bronchitic rats. We conclude that it is possible that an increase in sialyl transferase activity in a Golgi-containing fraction of bronchitic lungs could explain the relative increase in sialomucins in bronchitic subjects.     

Undifferentiated (embryonal) sarcoma of the liver: radiologic- pathologic correlation

Undifferentiated (embryonal) sarcoma (UES) is an uncommon malignant mesenchymal hepatic tumor that occurs in older children and young adults. Fourteen cases are correlated with radiologic and pathologic findings. Radiologic findings reflect the spectrum of solid to cystic appearances observed in the gross specimens. Sonography typically demonstrates a large mass that may be predominantly solid with many small anechoic spaces or may be cystic. Computed tomography reveals a hypodense mass with hyperdense septa of variable thickness and a dense peripheral rim corresponding to the fibrous pseudocapsule of the tumor. Angiographically, UES is usually hypovascular with tumoral vessels, although hypervascular and avascular patterns occur. Prognosis of UES is poor, with a median survival of less than 1 year. Radiologists should be familiar with this malignant tumor since it may present as a large cystic hepatic mass, suggestive of a benign lesion.     

Radioimmunoassay of factor V in human plasma and platelets

Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 micrograms/ml of plasma with an average value of 7.0 +/- 2.0 micrograms/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63-1.93 microgram of factor V is present per 2.5 X 10(8) platelets (4612-14128 molecules of the factor V platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.