Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Monoclonal antibody-defined antigens of human prostate cancer cell line PC3

Summary Over 600 hybridomas were derived from the immunization of mice with live cells and aqueous extracts of the human prostatic carcinoma cell line PC3. A total of 26 hybridomas with restricted reactivities were selected, subcloned and antibodies tested on a variety of tumor and normal cells. Seven monoclonal antibodies showed reactivity for prostate cancer and other tumor cell lines, including breast carcinomas. Three of the antibodies obtained after immunization with live cells reacted with live cells only and three of the four antibodies obtained after immunization with cell extract reacted with cell extracts and spent culture media. The fourth antibody in the latter group was reactive only in the immunoperoxidase staining assay. Antibody PrS5 recognized a 90,000 molecular weight molecule from ¹²⁵I-surface-labeled cells in immunoprecipitation analysis. Antibodies PrE3 and PrD8 detected a nonacid glycolipid pentasaccharide from PC3 cells and meconium, and a glycoprotein of 115,000 molecular weight from ¹²⁵I-surface-labeled red blood cells. The similar patterns of reactivity in RIAs and antigen analysis suggest that antibodies PrE3 and PrD8 recognize the same molecule. The results emphasize the usefulness of immunohistochemistry in the testing of monoclonal antibodies and the impact of the form in which the antigen is presented on the resultant antibody specificity     

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Colestipol-induced changes in LDL composition and metabolism. II. Studies in humans

We investigated the effect of the bile acid sequestrant, colestipol hydrochloride, on the composition and metabolism of human low density lipoprotein (LDL). Colestipol treatment produced a disproportionate decrease in LDL cholesterol compared to LDL apoB, resulting in a significant decrease in the LDL cholesterol/apoB ratio. Electron microscopy revealed that LDL particles were smaller in size and analytical ultracentrifugation demonstrated that colestipol therapy selectively depleted larger, more buoyant LDL particles of Sf degrees 6-7. Thus, colestipol therapy produced LDL that were smaller in size, more dense, and characterized by a decreased cholesterol to protein ratio. To determine whether the altered LDL had different metabolic properties, autologous LDL was isolated from subjects before and during colestipol therapy and their fractional catabolic rates (FCR) were then simultaneously determined in the same patient while on therapy. Eight LDL turnover studies comparing the catabolism of LDL isolated during therapy (Rx-LDL) and LDL isolated off therapy (Con-LDL) were performed in six subjects. All subjects responded to colestipol treatment, with an average 29% fall in LDL cholesterol. In four of six subjects, and in six of eight studies, the FCR of Rx-LDL was substantially slower than that of Con-LDL. These studies demonstrate that a drug intervention may alter subpopulations of LDL particles in such a way that overall LDL composition is changed. This alteration may independently affect the intrinsic metabolic behavior of the LDL. We suggest that such drug- (or dietary-) induced changes in LDL composition need to be considered in kinetic studies designed to assess the overall impact of the perturbation being studied.     

Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance.

The spin-labeling method was used to investigate human carbonic anhydrase, HCA II, undergoing unfolding induced by guanidine-HCI (Gu-HCI). The spin-probe, N-(2,2,5,5-tetramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzyme. The electron paramagnetic resonance spectrum of the folded structure showed the characteristic slow motional spectra. When the concentration of the denaturing agent, Gu-HCI, was gradually increased, new spectral components with narrower lines evolved to give complex electron paramagnetic resonance spectra, apparently containing superimposed contributions from several components of different mobility. By a differentiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCI concentration. The amplitude of difference spectra versus Gu-HCI concentration showed two distinct maxima, indicating the existence of a folding intermediate state/structure. The results were found to agree with optical absorption data, which showed similar transitions at the same Gu-HCI concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, folding intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCI were obtained by least squares fitting of the simulated spectra to the experimental ones. The folding intermediate was found to have a maximum population of 39 +/- 4% at approximately 0.7 M Gu-HCI.     

Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols.

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID50's for prostaglandin (PG) E2 synthesis in these cells were 0.1 muM for 2,6-xylenol, 5 muM for tricresol, 6 muM for p-cresol, 7 muM for o-cresol, 15 muM for 3,5-xylenol, 30 muM for m-cresol and 100 muM for phenol. The corresponding values for aspirin and indomethacin were 4 muM and 0.02 muM, respectively. The substituted phenols also inhibited serotinin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG2.