Gamma-interferon counteracts interleukin-1 alpha stimulated expression of urokinase-type plasminogen activator in human endothelial cells in vitro.

The effect of gamma-interferon (gamma-IFN) on the interleukin-1 alpha (IL-1 alpha) induced stimulation of urokinase-type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and in human umbilical vein endothelial cells (HUVEC) was investigated. When gamma-IFN and IL-1 alpha were added to the cells simultaneously, gamma-IFN inhibited the IL-1 alpha induced increase in u-PA antigen production in both HFMEC and HUVEC in a dose dependent fashion, with a maximum inhibitory effect achieved between 2.0 and 20.0 U/ml of gamma-IFN. Pretreatment of HFMEC with gamma-IFN for 1 hour before addition of IL-1 alpha resulted in a significant reduction in u-PA synthesis. However, when HFMEC were pretreated for 8 hours with gamma-IFN before the addition of IL-1 alpha the reduction in u-PA production was even more significant. When gamma-IFN was added to HFMEC 1 hour after IL-1 alpha, a significant inhibition in u-PA synthesis was seen. In contrast only a slight inhibition in IL-1 alpha induced u-PA production was seen when gamma-IFN was added to the cells 8 hours after IL-1 alpha. gamma-IFN also inhibited significantly the IL-1 alpha induced increase in u-PA specific mRNA in HUVEC and HFMEC.     

A cholecystokinin-metabolizing enzyme in rat intestine

Acid extracts of rat intestine contain a material which metabolizes cholecystokinin-33 (CCK-33) to CCK-12. Soybean trypsin inhibitor had little effect on CCK metabolism by the intestinal material. The molecular weight of the CCK-metabolizing activity, estimated by gel filtration, was 34,000. These data suggest rat intestine contains a nontrypsin CCK-metabolizing enzyme. Results from gel filtration also suggest that large CCK forms can be artifactually degraded to smaller ones during chromatography.     

Relatedness measured by oligonucleotide probe DNA fingerprints and an estimate of the mating system of Sea Lavender (Limonium carolinianum)

Using DNA fingerprint markers within species and populations of wild plants requires information on the relationship between fingerprint similarity and relatedness. We identified a hypervariable marker based on oliog(GATA)₄-hybridization of DpnII-cut genomic DNA from Sea Lavender (Limonium carolinianum). Banding patterns were somatically stable and highly variable among unrelated individuals. Band molecular-weight sizing errors (as a percent of band molecular weight) were estimated at 0.44%±0.003 within gels and 0.76%±0.964 between gels. Band sizing errors defined a 99% confidence bin of ±0.95% (1.90% total) of molecular weight. Band-sharing estimates were based on this bin size and on variance estimates that compensate for non-independent comparisons. Band-sharing among nine unrelated individuals () was 0.198±0.O11. Experimental pollinations designed to produce selfed, fulland half-sib progeny groups led to five selfed progeny groups and no outcrossed progeny (mean band-sharing, ovS=0.468±0.074). A linear regression between band-sharing (S) and relatedness (r) assuming 17% inbreeding was r=0.006+0.914*S (R²=0.973) and established the maximum amount of inbreeding. ovS(0.392±0.022) estimated from wild pollinated seeds from four maternal families was intermediate to unrelated individuals and experimental selfed progeny, giving evidence for mixed mating in wild plants. More extensive plant pedigrees with known levels of inbreeding will be needed to measure variation in the relationship between S and r among populations and families.     

13C MAS NMR studies of crystalline cholesterol and lipid mixtures modeling atherosclerotic plaques.

Cholesterol and cholesteryl esters are the predominant lipids of atherosclerotic plaques. To provide fundamental data for the quantitative study of plaque lipids in situ, crystalline cholesterol (CHOL) and CHOL/cholesteryl ester (CE) mixtures with other lipids were studied by solid-state nuclear magnetic resonance with magic-angle-sample spinning. Highly distinctive spectra for three different crystalline structures of CHOL were obtained. When CHOL crystals were mixed with isotropic CE oil, solubilized CHOL (approximately 13 mol % CHOL) was detected by characteristic resonances such as C5, C6, and C3; the excess crystalline CHOL (either anhydrous or monohydrate) remained in its original crystalline structure, without being affected by the coexisting CE. By use of 13C-enriched CHOL, the solubility of CHOL in the CE liquid-crystalline phase (approximately 8 mol %) was measured. When phosphatidylcholine was hydrated in presence of CHOL and CE, magic-angle-sampling nuclear magnetic resonance revealed liquid-crystalline CHOL/phosphatidylcholine multilayers with approximately an equal molar ratio of CHOL/phosphatidylcholine. Excess CHOL existed in the monohydrate crystalline form, and CE in separate oil or crystalline phases, depending on the temperature. The magic-angle-sampling nuclear magnetic resonance protocol for identifying different lipid phases was applied to intact (ex vivo) atherosclerotic plaques of cholesterol-fed rabbits. Liquid, liquid-crystalline, and solid phases of CE were characterized.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

The mating system and relative performance of selfed and outcrossed progeny in Arabis fecunda (Brassicaceae)

We estimated rates of self-fertilization and inbreeding depression in the hermaphroditic perennial Arabis fecunda. Crosses were made on mesh-bagged wild plants, yielding 12 plants producing self-, outcross-, and naturally fertilized seeds that were then grown in a greenhouse. Analysis of variance indicated aboveground dry weight at 160 days differed among pollination treatments, but mean seed weight, number of seeds per fruit, and days to germination did not. For dry weight, selfed progeny have 9.4% reduction and naturally pollinated progeny a 3.7% reduction compared to outcrossed progeny, suggesting a 39% selling rate in Arabis. Significant inbreeding depression and a mixed mating system are evidence that the deleterious effects of self-fertilization are maintained through high rates of mildly deleterious mutation. Significant maternal-parent-by-pollination-treatment interactions for mean seed weight and dry weight are consistent with inbreeding depression caused by deleterious recessives and varying past maternal inbreeding.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.