Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains

We have previously reported on the secretion of a family of high Mr plasminogen activators (PAs) by a human lung cancer cell line [Harvey et al. (1991) Biochim. Biophys. Acta 1078, 360-368]. We have now extended these studies to several human cancer cell lines and a human embryonic lung cell line. In the present study with HPL-SK-1 lung cancer, A431 epidermoid cancer, ovarian carcinoma, and embryonic lung cell lines, we show that the 900- and the 660-kDa PAs are disulfide-bonded multiprotein oligomeric complexes. They are functionally and immunologically related to human urinary PA (uPA). Their size and PA activity are not destroyed by strong denaturants such as 8 M urea or 2% sodium dodecyl sulfate (SDS), suggesting that the uPA moiety is covalently associated with the rest of the molecule. It is only under strong denaturing conditions with 1.4 M beta-mercaptoethanol and 2% SDS that the uPA moiety could be released as a 21- to 23-kDa fragment along with two major polypeptide chains of 70 and 40 kDa, respectively. The presence of the uPA active center in the reduced PA660 was demonstrated by [3H]diisopropylphosphorofluoridate labeling and by Western blot using a monoclonal antibody to uPA B chain. N-terminal amino acid sequencing of the 70- and 40-kDa polypeptides, respectively, showed homology to the neural cell adhesion molecule and the beta chain of haptoglobin. A minor fragment of 18 kDa obtained under strong reduction conditions was also sequenced and shown to share homology with the alpha chain of haptoglobin. Western blot analysis of the reduced PAs with monoclonal antibody to the neural cell adhesion molecule and rabbit anti-haptoglobin confirmed the homologies obtained by the sequence data. Further, immobilized monoclonal antibodies to the neural cell adhesion molecule, uPA B chain, and rabbit anti-haptoglobin bound the multiprotein complexes with uPA activity, from A431, ovarian cancer, and embryonic lung cell lines. The bound material, after dissociation, exhibited PA activity that was inhibited by monoclonal antibody to the uPA B chain. These data suggest that in tumor and embryonal cell lines, in addition to proper folding and assembly of proteins by intramolecular disulfide bond formation in the endomembrane compartment, intermolecular disulfide bonds could also occur, producing multiprotein oligomers as in the present case. Formation of such oligomers may have a selective advantage for such cells in the focalization of proteolytic activity through the interaction of the neural cell adhesion molecule domain with the extracellular matrix and in immunosuppression of lymphocytes by the haptoglobin portion of the complex.     

Production responses by early lactation cows to whole sunflower seed or tallow supplementation of a diet based on barley

A 2-yr study to evaluate the effectiveness of whole sunflower seed as a source of fat was conducted with 18 primiparous and 31 multiparous Holstein cows. The three diets evaluated were a basal diet based on barley (control), a basal diet supplemented with 2.7% tallow, and a basal diet supplemented with 7.1% whole sunflower seeds. The DMI of lactating cows during the 16-wk test period was not influenced by supplementation with either sunflower seeds or tallow. Milk production was 34.4, 34.6, and 35.5 kg/d for cows fed the control diet or the diets supplemented with sunflower or tallow, respectively, and was not influenced by diet. The production and concentrations of milk protein, fat, and SNF also were not influenced by diet. The concentrations of C6:0 to C14:1 fatty acids were highest in the milk of cows fed the control diet. The concentrations of C10:0 to C16:1 were higher when cows were fed the diet with the tallow supplement than when they were fed the diet with the sunflower supplement. However, the concentrations of C18:0 to C18:2 and C20:0 were higher in the milk of cows that were fed the sunflower supplement than in the milk of cows that were fed the tallow supplement or the control diet. Concentrations of individual VFA and the ratio of acetate to propionate were not influenced by diet. Body weight, body condition score, and reproduction parameters were similar for all diets, suggesting that there were no effects on subsequent production. The performance of cows fed whole sunflower seeds as a source of energy appeared to be similar to the performance of cows fed traditional high energy diets based on barley. The fatty acid profile of the milk of cows fed diets supplemented with sunflower seeds was more favorable than that of the milk of cows fed diets supplemented with tallow.     

Single molecule microscopy: peak-frequency trajectories and linewidth distribution

Fluorescence microscopy has been adapted to cryogenic temperatures and combined with efficient image processing and analysis to observe many single molecules in parallel. The capabilities of this setup are demonstrated with the parallel recording of peak-frequency trajectories for 62 terrylene-molecules in n-hexadecane and with the measurement of 918 single molecule linewidths for terrylene in dodecane in 200 s.     

Expression of human leukocyte antigens class I and class II on cultured biliary epithelial cells after cytomegalovirus infection

The influence of cytomegalovirus (CMV) infection on the HLA expression on cultured biliary epithelial cells (BEC) was investigated. CMV-infection augmented expression of HLA class I but not of HLA class II. CMV reduced the IFN-gamma-mediated induction of the de novo expression of HLA class II while the stimulated expression of HLA class I was not impaired. Autologous but not allogeneic PBL responded to CMV-infected BEC. This response resulted in upregulation of HLA class I on BEC which was significantly higher compared with the expression on infected BEC alone or on uninfected BEC cocultured with autologous PBL. The results suggest that CMV modulates the immunogenic potential of BEC, which is important for the HLA and CMV-mediated pathomechanisms in vivo.     

Embolic Doppler ultrasound signal detection using discrete wavelet transform

Asymptomatic circulating emboli can be detected by Doppler ultrasound. Embolic Doppler ultrasound signals are short duration transient like signals. The wavelet transform is an ideal method for analysis and detection of such signals by optimizing time-frequency resolution. We propose a detection system based on the discrete wavelet transform (DWT) and study some parameters, which might be useful for describing embolic signals (ES). We used a fast DWT algorithm based on the Daubechies eighth-order wavelet filters with eight scales. In order to evaluate feasibility of the DWT of ES, two independent data sets, each comprising of short segments containing an ES (N=100), artifact (N=100) or Doppler speckle (DS) (N=100), were used. After applying the DWT to the data, several parameters were evaluated. The threshold values used for both data sets were optimized using the first data set. While the DWT coefficients resulting from artifacts dominantly appear at the higher scales (five, six, seven, and eight), the DWT coefficients at the lower scales (one, two, three, and four) are mainly dominated by ES and DS. The DWT is able to filter out most of the artifacts inherently during the transform process. For the first data set, 98 out of 100 ES were detected as ES. For the second data set, 95 out of 100 ES were detected as ES when the same threshold values were used. The algorithm was also tested with a third data set comprising 202 normal ES; 198 signals were detected as ES.     

Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).