Differences in tetranectin immunoreactivity between benign and malignant breast tissue

Summary Tetranectin (TN) is a human, plasminogen kringle 4 binding plasma protein with ubiquitous cellular distribution and lectin-like characteristics. By means of the peroxidase-antiperoxidase staining technique a polyclonal and a monoclonal antibody were used to demonstrate TN within the intracellular as well as the extracellular compartment of invasive breast carcinoma. Whereas cell associated TN was universal showing only quantitative differences depending of the growth pattern of the tumor, 78 of 133 tumors displayed TN extracellularly as well. The occurrence of this stromal TN immunoreactivity was closely associated with desmoplasia, recognized morphologically by an increase in fibroblastic cells and immunohistochemically by an intense staining for the connective tissue glycoprotein fibronectin (FN). Benign breast tissue displayed a universal, intense cytoplasmic but no extracellular reaction for TN, with the exception of rare foci of granulation tissue and around dilated cysts. Functional studies have shown that human embryonal lung fibroblasts increase their release of TN to the growth medium upon stimulation. The presence of TN extracellularly within fibroblast-rich foci of desmoplasia (and granulation tissue) suggests that a similar increased release of the protein takes place in vivo during active states. Desmoplasia has been found to have a protective effect on tumor cell propagation and metastasis in a murine model. The molecular interactions, which are responsible for this effect, are undoubtedly complex. However, TN may, by its specific binding to kringle 4 of plasminogen and its high affinity for sulphated polysaccharides, add to the understanding of how plasminogen activation is modulated at the local extracellular level.     

Bleomycin-iron complexes and DNA radiation damage.

Radiation in the form of high-energy electrons dose-dependently activates bleomycin-Fe3+ in oxygen-containing DNA solutions. This activation causes a DNA fragmentation and a release of oxidative degradation products from the DNA. During irradiation, bleomycin-chelated Fe3+ is reduced to Fe2+. The kinetics of DNA base propenal-formation (measured by reaction with thiobarbituric acid) and iron-reduction (measured by bathophenanthroline chelation) are similar, with a yield of 1 mol base propenal/6 mol Fe3+ reduced. The activation of bleomycin-Fe3+ by irradiation could be instrumental in the synergistic action of radiotherapy and bleomycin observed on simultaneous administration in vivo.     

A high-pressure form of sulfuric acid monohydrate as determined by X-ray and neutron diffraction

Two high-pressure polymorphs of sulfuric acid monohydrate (oxonium hydrogensulfate) have been obtained at ambient temperature by crystallisation at high pressure from the liquid at 1.3 GPa (form III) and by direct compression of the ambient-pressure form I first to 1.26 GPa (form II) and then to 1.72 GPa (form III). The structure of form III was solved by single crystal X-ray diffraction and this structure was used as the basis for the refinement of hydrogen positions using high-pressure neutron powder diffraction data. Form III crystallises in the orthorhombic crystal system at 1.97 GPa, and features parallel chains of hydrogensulfate ions linked by oxonium ions to form a three-dimensional hydrogen-bonded network. On further compression to 3.05 GPa, the direction of maximum compressibility is found to be along the a-axis and is associated with the shortening of a hydrogen bond between a hydrogensulfate ion and an oxonium ion. The structure of form II remains elusive although at ambient temperature it is stable (or metastable) at pressures as low as 0.42 GPa, perhaps indicating that it could be recoverable to ambient-pressure at low temperature.     

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.     

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Competition for Space Is Controlled by Apoptosis-Induced Change of Local Epithelial Topology

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Discovery of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu₅ negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu₅ NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu₅ assay, and an exemplar analog 27 was >60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.