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A pronase resistant 85-kd glycoprotein in the Concanavalin A-binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-85.

Purpose: Oocyte donation is a well-established method of assisted reproduction for women with irreversible infertility and with previous implantation failures after in vitro fertilization. Although the pregnancy rates are very high, sometimes implantation does not occur even after various attempts. We report the first two cases of transfer of zona-free blastocysts in oocyte donation cycles that developed to normal pregnancies and births., Methods: The patients had undergone three previous standard oocyte donation cycles with failure of implantation. Endometrium preparation was performed after suppression of the pituitary function, with E2 valerate and Progesterone at the day of oocyte retrieval. Normally fertilized embryos were cultured in Earle's culture medium until Day 3 and in S2 medium until Day 5. For each patient, the zonae of two fully expanded blastocysts were enzymatically removed with 0.5% pronase. Zona-free blastocysts were transferred for the patients 2 h later., Results: On Day 12 after transfer, pregnancies were confirmed with elevated serum levels of beta hCG. A gestational sac with a foetal heart beat was seen by ultrasound 15 days later, in each patient. Normal healthy babies were born at 38 and 39 weeks of pregnancy., Conclusions: This is the first report of successful pregnancies and births after oocyte donation and transfer of zona-free blastocysts in human. It not only shows the feasibility of the treatment but also opens a new alternative for the patients with repetitive implantation failure after OD cycles.

1. The hyperpolarization-activated If current was recorded in inside-out macropatches from sino-atrial (SA) node myocytes during exposure of their intracellular side to pronase, in an attempt to verify if cytoplasmic f-channel domains are involved in both voltage- and cAMP-dependent gating. 2. Superfusion with pronase caused a quick, dramatic acceleration of channel opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cleavage of channel regions which contribute to gating. 3. If recorded from patches normally responding to cAMP became totally insensitive to cAMP following pronase treatment, indicating partial or total removal of channel regions involved in the cAMP-dependent activation. 4. The fully activated I-V relationship was not modified by pronase, indicating that internal proteolysis did not affect the f-channel conductance. 5. The changes in If kinetics induced by pronase were due to a large depolarizing shift of the f-channel open probability curve (56.5 +/- 1.1 mV, n = 7). 6. These results are consistent with the hypothesis that cytoplasmic f-channel regions are implicated in dual voltage- and cAMP-dependent gating; also, since pronase does not abolish hyperpolarization-activated opening, an intrinsic voltage-dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thus includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.

Fibrin deposition and leukocyte accumulation are classic histopathologic hallmarks of acute and chronic inflammation. Although both of these processes are common to inflammatory processes, surprisingly little is known about the interrelationship between fibrin deposition and leukocyte accumulation. We hypothesized that: 1) fibrin can regulate inflammation by directly inducing leukocyte chemotactic factor (LCF) expression from vascular endothelial cells (VEC), and 2) this fibrin-induced VEC expression of LCF would enhance leukocyte recruitment seen in acute and chronic inflammation. To test this hypothesis, we developed an in vitro model system of in situ polymerization of fibrin on isolated bovine pulmonary artery endothelial cells in culture. In the initial phase of our studies, we found that fibrin induced a time- and dose-dependent expression of leukocyte migration activity from VEC. Checkerboard analysis demonstrated that the leukocyte migration activity present in the fibrin-stimulated VEC culture supernatant was truly chemotactic in nature. In an effort to begin to characterize this chemotactic activity, we demonstrated that this fibrin-induced LCF activity was 1) protein dependent, 2) not derived from fibrin(ogen), and 3) had an apparent m.w. of 6 to 12 kDa. Our further studies demonstrated that fibrin was a potent stimulus for IL-8 Ag expression from the VEC. Thus, these studies clearly demonstrate that fibrin has the capability to induce leukocyte chemotactic activity in general, and IL-8 activity specifically, from VEC. These studies support our hypothesis that fibrin is an important activator of vascular endothelial cells in vivo.

In mice, active protection against Pseudomonas aeruginosa could be induced with two fractions derived from a crude preparation of ribosomes from P. aeruginosa. The two fractions were obtained by gel filtration chromatography of the crude ribosomal preparation on Sepharose CL-2B. In fraction I, less than 1% of the ribonucleic acid (RNA) applied to the column was recovered. Fraction II contained RNA and protein in a ratio of 1.94. The presence of ribosomes in this fraction was confirmed by analysis on a sucrose density gradient. The protection by fraction I was not affected by treatment with ribonuclease; in contrast, incubation of fraction II with ribonuclease completely abolished active protection. Fraction I contained lipopolysaccharide (LPS) as was indicated by the presence of 2-keto-3-deoxyoctonic acid. No LPS was found in fraction II. The adjuvant dimethyl dioctadecyl ammonium bromide enhanced the protection by fraction II; however, immunity by a low dose of fraction I was abolished by dimethyl dioctadecyl ammonium bromide. Protection by fractions I and II appeared to be restricted to the homologous serotype of P. aeruginosa. These results indicate that RNA is required for protection by fraction II. Active protection by fraction I is likely due to LPS.