Purification of human albumin by the combination of the method of Cohn with liquid chromatography

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60 degrees C for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 +/- 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were < or = 5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.     

DNA fiber fluorescence in situ hybridization analysis of immunoglobulin class switching in B-cell neoplasia: aberrant CH gene rearrangements in follicle center-cell lymphoma

Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cmu-Cdelta genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cgamma gene or pseudogene in the 3' cluster.     

Fibroblast variants nonresponsive to fibroblast growth factor 1 are defective in its nuclear translocation

Fibroblast growth factors (FGF) elicit biological effects by binding to high affinity cell-surface receptors and activation of receptor tyrosine kinase. We previously reported that two NIH/3T3 derivatives, NR31 and NR33 (NR cells), express high levels of full-length FGF-1 and exhibit a complete spectrum of transformed phenotype. In the present study, we report that NR cells respond to the mitogenic stimulation of truncated FGF-1 but not to the full-length FGF-1. Incubation of the NR cells with either form of FGF-1 resulted in its binding to cell-surface FGF receptors, activation of mitogen-activated protein (MAP) kinase, and induction of c-fos and c-myc. These data demonstrate that the FGF receptor-mediated, MAP kinase-dependent signaling pathway is not defective in the NR cells. Our data further suggest that the activation of MAP kinase in response to full-length FGF-1 is not sufficient for mitogenesis. Subcellular distribution of exogenously added FGF-1 demonstrated that full-length FGF-1 fails to translocate to the nuclei of NR31 cells. Although the full-length FGF-1 was detected in the nuclear fractions of both NIH/3T3 and NR33 cells, its half-life is much shortened in NR33 than in NIH/3T3 cells. These observations suggest that non-responsiveness of the two NR cell lines may be due to defectiveness at different steps of nuclear translocation mechanism of FGF-1.     

Identification of partial disjunction, parity, and threshold functions

Let F be a class of functions obtained by replacing some inputs of a Boolean function of a fixed type with some constants. The problem considered in this paper, which is called attribute efficient learning, is to identify “efficiently” a Boolean function g out of F by asking for the value of g at chosen inputs, where “efficiency” is measured in terms of the number of essential variables. We study the query complexity of attribute-efficient learning for three function classes that are, respectively, obtained from disjunction, parity, and threshold functions. In many cases, we obtain almost optimal upper and lower bound on the number of queries.     

Amplification of the c-myc gene in human hepatocellular carcinoma: biologic significance

To elucidate the prevalence and biologic significance of the c-myc gene in human hepatocellular carcinoma (HCC), DNA samples were taken from the paired tumorous and nontumorous tissues of 77 cases of resected primary HCC and were analyzed by Southern blot hybridization. We demonstrated modest, but significant c-myc amplification (group A) in 28 (36.4%) of the cases: 1.6- to 2.0-fold in 18, 2.1- to 3.0-fold in four, and > 3.0-fold in six. Compared to HCC without c-myc amplification (group B), group A HCC occurred more often in patients < 50 years old (54.5% vs 29.1%, p < 0.02) with serum alpha-fetoprotein (AFP) levels > 320 ng/mL (61.1% vs 14.6%, p < 0.00002). Group A HCC occurred more frequently in patients with hepatitis B virus infection than in those with hepatitis C virus infection (p < 0.03). Group A HCC was more likely to be poorly differentiated (44.8% vs 10.5%, p < 0.004) and associated with intrahepatic portal vein spread (57.1% vs 28.6%, p < 0.02). The c-myc amplification did not correlate with sex or tumor size. For small HCC, group A had a worse one-year survival rate than group B (72.2% vs 90.9%, p < 0.04). These findings suggest that c-myc amplification is not an uncommon event in human hepatocarcinogenesis, occurs more frequently in young patients who have an elevated serum AFP level or HBV infection, and is related to the biologic behavior of HCC.     

Comparison of cannabinoid binding sites in guinea-pig forebrain and small intestine

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity. [3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%). Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg(-1)) than in intestinal membranes (2092 fmol mg(-1)). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively). The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg(-1)) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg(-1)). O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940). We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that 0-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation.     

Zeitschriftenreferate

Ohne Zusammenfassung     

Nonsustained ventricular tachycardia in severe heart failure. Independent marker of increased mortality due to sudden death. GESICA-GEMA Investigators

BACKGROUND: The goal of the study was to determine the prognostic value of nonsustained ventricular tachycardia (NSVT) in total mortality in severe congestive heart failure (CHF) and in death modes. NSVT is associated with an increased mortality in CHF. However, the predictive value of NSVT as a marker for sudden death or death due to progressive heart failure has not been determined. METHODS AND RESULTS: Five hundred sixteen patients from the GESICA trial (33.4% with NSVT) were initially studied with the results of 24-hour Holter and 2 years of follow-up. Within 2 years, 87 of 173 patients (50.3%) with NSVT and 106 of 343 patients (30.9%) without NSVT died. Relative risk (RR) was 1.69 (95% confidence interval [CI], 1.27 to 2.24; P < .0002), and Cox proportional hazard analysis was 1.62 (95% CI, 1.22 to 2.16; P < .001). Sudden death increased from 8.7% (30 of 343) to 23.7% (41 of 173) in patients with NSVT (RR, 2.77; 95% CI, 1.78 to 4.44; P < .001). Progressive heart failure death was also increased from 17.5% (60 of 343) to 20.8% (36 of 173) (P = .22). Quantitative analysis of 24-hour Holter (first 295 patients) demonstrated that couplets had a similar RR to that of NSVT for both total mortality (RR, 1.81; 95% CI, 1.22 to 2.66; P < .002) and sudden death (RR, 3.37; 95% CI, 1.57 to 7.25; P < .0005). Couplets and/or NSVT (ventricular repetitive beats) were even more predictive for sudden death (RR, 10.1; 95% CI, 1.91 to 52.7; P < .01). CONCLUSIONS: In patients with CHF, NSVT is an independent marker for increased overall mortality rate and sudden death. The absence of NSVT and ventricular repetitive beats in a 24-hour Holter indicates a low probability of sudden death.