Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

Relationship between computerized morphonuclear image analysis and histopathologic grading of breast cancer

A pilot study analyzed the relationship between several morphonuclear parameters and the Bloom-Richardson score for 37 invasive, not-otherwise-specified (NOS) ductal breast carcinomas. The SAM-BA 200 cell image processor and its software were used to measure the nuclear features on Feulgen-stained imprint smears. Two parameters representing the numbers of large and dense chromatin clots and two parameters describing the heterogeneity of the chromatin among nuclei in a specimen evolved in a continuous manner parallel with the Bloom-Richardson score from stages NOS-4 to NOS-8. The systematic measurement of these four parameters on a large series of breast cancers may be able to define an objective and reproducible "scale" of differentiation that could be a helpful tool for pathologists and clinicians.     

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme prosthetic group required for acetylation of prostaglandin H synthase by aspirin

The ability of aspirin to acetylate PGH synthase was determined by reacting [3H-acetyl]-aspirin with purified enzyme followed by high pressure liquid chromatography analysis of the protein components of the reaction mixture. Heme-reconstituted enzyme incorporated approximately one acetyl group per 70-kDa subunit, whereas apoprotein incorporated 0.1 acetyl group per subunit. The ability of the heme prosthetic group to enhance acetylation of the protein was correlated with its ability to protect the Arg253-Gly254 peptide bond from cleavage by trypsin. Thus, heme-induced alteration of protein conformation may contribute to the enhanced labeling of Ser506 by aspirin. The present results indicate that irreversible inactivation of prostaglandin H synthase by aspirin occurs only when the heme prosthetic group is bound to the protein. Considering its short in vivo half-life, it is likely that aspirin inactivates only the steady-state fraction of PGH synthase in a cell that is active but not newly synthesized apoprotein. This may contribute to the differential kinetics of inactivation and recovery of PGH synthase activity in platelets and vascular endothelial cells after administration of low dose aspirin as a prophylactic agent against cardiovascular disease.     

Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa

Summary The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies.Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.Abbreviations HGL Human gastric lipase - SDS PAGE Sodium dodecyl sulfate-polyAcrylamid gel electrophoresis - PBS Phosphate buffer saline     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Comitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways

We have recently developed a mAb, anti-1F7, which defines a family of structures found to include the molecule recognized by anti-Ta1 (CD26). In this paper, we demonstrated that binding of 1F7 by solid-phase immobilized anti-1F7 mAb but not anti-Ta1 mAb has a comitogenic effect by inducing proliferation of human CD4+ T lymphocytes in conjunction with submitogenic doses of anti-CD3 or anti-CD2. The proliferative response induced via the CD3-1F7 or CD2-1F7 pathways is associated with the IL-2 autocrine pathway, including IL-2 production. IL-2R expression and anti-IL-2R (Tac) inhibition. Furthermore, solid-phase immobilization of anti-1F7 but not anti-Ta1 acts in conjunction with submitogenic doses of PMA to mediate a comitogenic effect in the absence of anti-CD3 or anti-CD2, leading to CD4+ T cell proliferation. PMA treatment, in the meantime, leads to enhanced expression of 1F7 on the T cell surface. Despite its functional association with both pathways of activation, however, the 1F7 structure is not comodulated with the CD3/TCR complex nor the CD2 molecule. These findings thus suggest that the CD26 Ag is involved in CD3 and CD2-induced human CD4+ T cell activation.     

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.     

Praziquantel-induced secretion of proteolytic enzyme from Schistosoma mansoni cercariae

The effect of praziquantel (PZQ) on secretion of proteolytic enzyme by Schistosoma mansoni cercariae was examined using an azocoll assay. The cercariae secreted proteolytic enzyme in various concentrations of PZQ (0.1, 1, and 10 micrograms/ml), but secretion of enzyme was highest at the lowest concentration. PZQ-induced secretion of proteolytic enzyme was partially inhibited by treatment with verapamil and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetra-acetic acid but not by calmodulin antagonist W-7 and protein kinase C inhibitor H-7.