Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Effects of various compounds on histidine decarboxylase activity: Active site mapping

The effect of about one hundred compounds on the activity of histidine decarboxylase partially purified from whole bodies of fetal rats was determined. Most of them at their 10 mM concentration had little effect on the enzyme activity; but 12 compounds inhibited the enzyme to a greater extent than 30%. Among these, except for -methylhistidine that has been known to be a strong and specific inhibitor, DOPA, homocysteine, cysteine, methionine and urocanic acid were the best inhibitors; -phenyllactic acid, phenylpyruvic acid and carnosine were less strong inhibitors; valine, oxaloacetic acid andN -methylimidazole acetic acid were weak inhibitors. Histamine had no inhibitory action. Thus, the substrate binding site of histidine decarboxylase is very rigid and specific forl-histidine.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Topical application of FK506 (tacrolimus) ointment inhibits mite antigen-induced dermatitis by local action in NC/Nga mice

BACKGROUND: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. METHODS: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. RESULTS: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs. CONCLUSIONS: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.     

Simultaneous localization of histamine and factor VIII-related antigen in the endothelium of the human umbilical vein

Simultaneous immunoelectron microscopic localization of histamine and factor VII-related antigen was examined on the same ultrathin section of the endothelium of the human umbilical vein from full-term deliveries by means of the double-immunolabeling technique. Small gold particles demonstrating antibody reaction with histamine are preferentially located in the cytoplasmic matrix and organelles, especially in mitochondria and on the luminal membrane surface of the endothelial cells. The gold particles representing histamine immunoreactivity also located on some of Weibel Palade (WP) bodies. In contrast, large gold particles demonstrating factor VII-related antigen are concentrated preferentially on most WP bodies. Single labeling of either histamine or factor VIII-related antigen shows similar results to those of the double labeling. The present study indicates that some WP bodies are involved in storage of both factor VIII-related antigen and histamine, but others store factor VIII-related antigen only. This difference in contents of WP bodies may be induced during the development and maturation process of this inclusion. At any rate, it is reasonable to consider that WP bodies have important roles in both vascular tonus and hemostasis during the vascular obliteration.     

The Serum Factor from Patients with Ulcerative Colitis that Induces T Cell Proliferation in the Mouse Thymus Is Interleukin-7

The disturbance of immune regulatory T cells is related to the pathogenesis of ulcerative colitis. Here we demonstrated and characterized the serum factor from ulcerative colitis patients that induced proliferation of intrathymic T cells. The factor isolated from the patient sera by a combination of gel filtration and anion-exchange chromatography induced proliferation of CD4⁺CD8^– intrathymic T cells in the organ-cultured embryonic mouse thymus. Purification and amino acid sequence analysis of the serum factor demonstrated that the N-terminal 12 sequence was homologous to that of interleukin-7. SDS-PAGE and Western blot confirmed that purified serum factor was interleukin-7. Enzyme immunoassay demonstrated that the serum interleukin-7 concentration was significantly increased in the patients. PCR and Southern blot hybridization demonstrated that interleukin-7 mRNA expression was increased in the thymus tissues from patients but decreased in the colonic mucosa. Since interleukin-7 is a crucial cytokine for proliferation and differentiation of T cells in the thymus, the present study indicates that interleukin-7 may contribute to the disturbance of immune regulatory T cells in ulcerative colitis.     

Three-dimensional analysis of alveolar structure in usual interstitial pneumonia

The etiology of usual interstitial pneumonia (UIP), a progressive lung disease, remains unclear. We examined alveolar structure in UIP three-dimensionally. Lung biopsy specimens from five patients with idiopathic pulmonary fibrosis were used. Sections 150-microm thick were stained with elastica solution for elastic fibers, with alpha-smooth muscle actin antibody for myofibroblasts, with anti-Thomsen-Friedenreich antibody for type-II pneumocytes and with anti-CD34 antibody for blood vessels. We examined them three-dimensionally using a laser confocal microscope or light microscope. In the fibrotic lesions, the thick elastic fibers forming the alveolar framework were not particularly dense considering the reduction in alveolar volume. Near the fibrotic lesions, some of the thin elastic fibers in the alveolar wall were slightly sinuous and ended with rounded tips. Type-II pneumocytes had proliferated and were distributed uniformly over the alveolar surface. Smooth muscle actin filaments were detected only around the alveolar orifice. These findings show that in UIP destruction of the elastic fiber framework of the alveoli may lead to irreversible focal alveolar collapse after damage to the alveolar epithelial cells, and proliferation of type-II pneumocytes may be involved with this elastolysis.     

Ring-opening-closing alternating copolymerization of 2-methyl-2-oxazoline with N-methyldiacrylamide

This paper describes a new ring-opening-closing alternating copolymerization (ROCAC) of 2-methyl-2-oxazoline (five-membered cyclic imino ether, 1 ) with N-methyldiacrylamide ( 2 ). The reaction of a 1 : 1 monomer feed ratio proceeded without any added catalyst to give an alternating copolymer 3 having two structural units formed by ring-opening and ring-closing (cyclization). The structure of copolymer 3 was determined by ¹H, ¹³C NMR, and IR spectroscopies. The extent of cyclization was at most 65%. The copolymerization was reasonably explained by a mechanism of propagation via zwitterion intermediates.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein-protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)(2), significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)(2) expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.