Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients

Ascites from ovarian cancer patients contain potent growth-promoting activity toward human ovarian cancer cells both in vitro and in vivo. This activity is associated with rapid increases in cytosolic free calcium ([Ca2+]i) as a consequence of phosphoinositide hydrolysis. In this study, we describe the purification, characterization, and identification of an ovarian cancer activating factor (OCAF) from ascites of ovarian cancer patients. We have isolated OCAF by a combination of solvent extraction, silica gel chromatography, and TLC. Mass spectral analysis, phospholipase sensitivity, and gas chromatographic behavior of purified OCAF indicate that OCAF is composed of various species of lysophosphatidic acid (LPA), including LPAs with polyunsaturated fatty acyl chains (linoleic, arachidonic, and docosahexaenoic acids). However, OCAF is more potent than sn-1 palmitoyl, oleoyl, or stearoyl LPA in increasing [Ca2+]i in ovarian cancer cells. The ability of OCAF to alter [Ca2+]i is sensitive to the effects of lipoxidase, whereas the activity of sn-1 oleoyl, stearoyl, or palmitoyl LPA is not, suggesting that polyunsaturated bonds in the fatty acyl chain of OCAF may account for its increased ability to activate ovarian cancer cells. Furthermore, a sn-2 linoleoyl LPA generated by phospholipase A1 treatment of synthetic phosphatidic acid is much more active than are sn-1 palmitoyl, stearoyl, or oleoyl LPA in increasing [Ca2+]i in ovarian cancer cells. Taken together, these data suggest that the ability of OCAF to increase cellular calcium may reside in the structure and/or location of the fatty acyl chain of LPA. Purified OCAF, at concentrations similar to those present in ascites from ovarian cancer patients, was sufficient to induce proliferation of ovarian cancer cells, as indicated by thymidine incorporation, reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, or colony formation. However, even at optimal concentrations of OCAF, proliferation was lower than that induced by FCS or ascites from ovarian cancer patients, indicating that, although OCAF may be a major regulator of ovarian cancer cells in vivo, it is not the sole mediator present in ascites, and it likely functions in concert with other growth factor activities.     

13C-urea breath test in Helicobacter pylori diagnosis and eradication. Correlation to histology, origin of ''false'' results, and influence of food intake

Obliterative or constrictive bronchiolitis is characterized by narrowing of the small airways, due to submucosal and peribronchiolar fibrosis, with chronic obstruction. The vast majority of cases of bronchiolitis obliterans are associated with other diseases and only few cases are idiopathic. We report on the main computed tomography (CT) methods used study obliterative bronchiolitis, the CT findings and the differential diagnosis with other diseases. The dynamic study of alveolar ventilation with CT uses inspiratory and expiratory CT or high-resolution CT (HRCT), spiral dynamic CT or HRCT with advanced image display, ultrafast CT. In abnormal cases HRCT shows direct and indirect signs of small airways disease. The most common (> 80%) sign of obliterative bronchiolitis is the so-called mosaic oligohemia, with low attenuating lobules, caused by air trapping and best seen on expiratory CT, associated with blood flow redistribution to more normal lobules; this finding simulates the ground-glass pattern from infiltrative lung disease. Differential diagnosis is more difficult in the presence of true ground-glass patterns associated with diffuse bronchiolar obstruction and also with mosaic oligohemia due to pulmonary vascular disease and pulmonary emphysema. HRCT can distinguish these diseases and dynamic CT is more sensitive than functional tests in detecting regional abnormalities and air trapping. The combination of HRCT, rapid volumetric scanning and advanced image display is a powerful tool study the normal and abnormal features of bronchiolar function and alveolar ventilation.     

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

The stimulatory effect of phytin added to skim milk on acid production of Lactobacillus casei was examined. Phytin stimulated acid production of L. casei fairly well. The stimulatory effect of phytin on acid production was not shown when phytin was treated with Dowex 50 (H+) and neutralized by NaOH solution. The incinerated product of phytin maintained almost equal stimulatory effect on acid production as that before processing. The addition of Mn2+ in the amount contained in a reagent phytin augmented the stimulatory effect on acid production markedly. The further addition of Fe3+, Ca2+, Mg2+ and PO4(3-) in amounts corresponding to their contents in the preparation of phytin as well as Mn2+ increased the effect slightly. The four preparations of phytin contained 0.045-0.20% of Mn, and the greater the Mn content was, the greater the potentiation of acid production.     

Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.