Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.     

Nitrosylated high density lipoprotein is recognized by a scavenger receptor in rat liver

In order to assess the presence of specific recognition sites for high density lipoprotein (HDL) in vivo, HDL was nitrosylated with tetranitromethane and the decay and liver uptake were compared with that of native HDL. The association of intravenously injected nitrosylated HDL (TNM-HDL) with liver was greatly increased as compared to native HDL. Using a cold cell isolation method, it became evident that the liver endothelial cells were responsible for the increased uptake of the modified HDL. The involvement of the endothelial cells in the uptake of TNM-HDL from the circulation could also be demonstrated morphologically by using the fluorescent dye dioctadecyl-tetramethyl-indocarbocyanine perchlorate (Dil) to label HDL. In vitro competition studies with isolated liver endothelial cells indicated that unlabeled modified HDL and acetylated LDL displaced iodine-labeled TNM-HDL, while no competition was seen with LDL and a slight displacement was seen with unlabeled native HDL. Nonlipoprotein competitors of the scavenger receptor such as fucoidin and polyinosinic acid blocked the interaction of TNM-HDL with the liver endothelial cells. Also the degradation of TNM-HDL was blocked by low concentrations of chloroquine. It can be concluded that a scavenger receptor on liver endothelial cells is involved in the clearance of tetranitromethane-modified HDL, which excludes the possibility of using TNM-HDL in vivo to assess the non-receptor-dependent uptake of HDL. The use of nitrosylated HDL in vitro as a low affinity control is limited to cell types that do not possess scavenger receptors, because cell types with scavenger receptors will recognize and internalize TNM-HDL by a high affinity scavenger pathway.     

Muscle architecture and fibre characteristics of rat gastrocnemius and semimembranosus muscles during isometric contractions

Rat gastrocnemius medialis (GM) and semimembranosus (SM) muscles have a very different morphology. GM is a very pennate muscle, combining relatively short muscle fibre length with sizable fibre angles and long muscle and aponeurosis lengths. SM is a more parallel-fibred muscle, combining a relatively long fibre length with a small fibre angle and short aponeurosis length. The mechanisms of fibre shortening as well as angle increase are operational in GM as well as SM. However, as a consequence of isometric contraction, changes of fibre length and angle are greater for GM than for SM at any relative muscle length. These differences are particularly notable at short muscle lengths: at 80% of optimum muscle length, fibre length changes of approximately 30% are coupled to fibre angle changes of 15 degrees in GM, while for SM these changes are 4% and 0.6 degrees, respectively. A considerable difference was found for normalized active slack muscle length (GM approximately 80 and SM approximately 45%). This is explained by differences of degree of pennation as well as factors related to differences found for estimated fibre length-force characteristics. Estimated normalized active fibre slack length was considerably smaller for SM than for GM (approximately 40 and 60%, respectively). The most likely explanation of these findings are differences of distribution of optimum fibre lengths, possibly in combination with differences of myofilament lengths and/or fibre length distributions.     

Survival of patients with malignancy-associated effusions

For a better understanding of the prognosis after the onset of a malignancy-associated effusion in patients known or subsequently shown to have cancer, survival time was compared with the findings and the date of the first cytologic diagnosis of an effusion. The number of patients studied was 254; 171 had a pleural and 83 a peritoneal effusion. The average survival time was 25.5 weeks, which was about equal for both sites of effusions. After two years, only 6% of all patients were alive. When the cytologic diagnosis of the effusion was "malignant," only 4% survived after two years; when the cytologic diagnosis was "suspicious for malignancy" and "nonmalignant," these figures were 5% and 7%, respectively. This indicates that a cytologic diagnosis of benign or nonmalignant is not a good indicator of a better prognosis in cancer patients for whom benign causes of the effusion have been excluded. There appeared to be a prognostic relationship between the length of the interval from the initial diagnosis of cancer to the time of examination of the first sample of the effusion: a longer interval was correlated with a better survival. When survival time was viewed in relation to therapy, patients whose pleural effusions were only treated by aspiration were found to have a particularly short average survival (13.9 weeks).     

Linkage analysis of families with hereditary retinoblastoma: nonpenetrance of mutation, revealed by combined use of markers within and flanking the RB1 gene.

Nonpenetrance of the inherited mutation responsible for retinoblastoma has been reported. By DNA analysis in families with hereditary retinoblastoma, it is possible to identify healthy individuals in whom the mutation is nonpenetrant. This requires the use of DNA markers both within and flanking the retinoblastoma gene. We have analyzed the segregation of several markers in 19 families (69 meioses) with hereditary retinoblastoma. In two families a carrier was identified who showed nonpenetrance of the mutation predisposing to retinoblastoma. The intragenic markers were informative in 15 pedigrees. The use of flanking markers from the same chromosomal region caused an increase of the number of informative families to 18. No crossing-over within the gene was observed. In one family an inherited deletion involving one of the RB1 alleles was detected. Our findings emphasize the use of a combination of both intragenic and flanking markers to obtain both the highest reliability of carrier detection in families with hereditary retinoblastoma and an accurate estimate of the frequency of nonpenetrance.     

Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.     

High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.