Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo.

Epithelial cell survival is dependent on extracellular signals provided by both soluble factors and by adhesion. In the mammary gland, extensive apoptosis of epithelial cells occurs rapidly when lactation ceases, but the mechanism of apoptosis induction is not known. In tissue culture, mammary epithelial cells require laminin as a survival ligand and specific beta1 integrins are necessary to suppress apoptosis. To explore the possibility that dynamic changes in cell-matrix interactions contribute to the onset of apoptosis during mammary involution in vivo, a detailed immunohistochemical analysis of the expression of integrin subunits and their extracellular matrix ligands during mouse mammary gland development has been performed. The kinetics of apoptosis were determined by using tissue samples obtained from virgin, pregnant, lactating, and involuting gland. The maximal elevation of apoptosis occurred within 24 hr of weaning as determined by histologic analysis and caspase-3 staining. A wide variety of laminin subunits, together with nidogen-1 and -2, and perlecan were identified within the basement membrane region of epithelial ducts, lobules, and alveoli in both human and mouse mammary gland. However, no change in the distribution of any of the basement membrane proteins or their cognate integrin receptors was observed during the transition from lactation to apoptosis. Instead, we discovered that altered ligand-binding conformation of the beta1 integrin to a nonbinding state coincided with the immediate onset of mammary apoptosis. This finding may provide a novel dynamic mechanism for inhibiting the transduction of extracellular matrix survival signals, thereby contributing to the onset of apoptosis in a developmental context in vivo.     

Mild atopic dermatitis lacks systemic inflammation and shows reduced nonlesional skin abnormalities

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Pre-clinical Study of <Superscript>213</Superscript>Bi Labeled PAI2 for the Control of Micrometastatic Pancreatic Cancer

Purpose: The urokinase plasminogen activator (uPA) and its receptor (uPAR) are expressed by pancreatic cancer cells and can be targeted by the plasminogen activator inhibitor type 2 (PAI2). We have labeled PAI2 with ²¹³Bi to form the alpha conjugate (AC), and have studied its in vitro cytotoxicity and in vivo efficacy. Methods and Materials: The expression of uPA/uPAR on pancreatic cell lines, human pancreatic cancer tissues, lymph node metastases, and mouse xenografts were detected by immunohistochemistry, confocal microscopy, and flow cytometry. Cytotoxicity was assessed by the MTS and TUNEL assay. At 2 days post-cancer cell subcutaneous inoculation, mice were injected with AC by local or systemic injection. Results: uPA/uPAR is strongly expressed on pancreatic cancer cell lines and cancer tissues. The AC can target and kill cancer cells in vitro in a concentration-dependent fashion. Some 90% of TUNEL positive cells were found after incubation with 1.2 MBq/ml of AC. A single local injection of ~222 MBq/kg 2 days post-cell inoculation can completely inhibit tumor growth over 12 weeks, and an intraperitoneal injection of 111 MBq/kg causes significant tumor growth delay. Conclusions: ²¹³Bi-PAI2 can specifically target pancreatic cancer cells in vitro and inhibit tumor growth in vivo. ²¹³Bi-PAI2 may be a useful agent for the treatment of post-surgical pancreatic cancer patients with minimum residual disease.     

Effects of subacute treatment with toluene on cerebrocortical alpha- and beta-adrenergic receptors in the rat. Evidence for an increased number and a reduced affinity of beta-adrenergic receptors

Subacute treatment with toluene (80-1500 p.p.m.) produces a dose-dependent reduction of affinity and increase in density of the beta-adrenergic antagonist [3H]dihydroalprenolol binding sites in the frontoparietal cortex of the male rat, while the binding characteristics of alpha 1-adrenergic ([3H]WB 4101) and alpha 2-adrenergic ([3H]p-aminoclonidine) binding sites in the same region is unaffected by this treatment as evaluated in vitro. Therefore, it is suggested that the cortical beta-adrenergic receptors are particularly vulnerable to the action of toluene in vivo. It is speculated that as a result cortical beta-adrenergic neurotransmission may be altered following exposure to low concentrations of toluene, possibly related to the physico-chemical properties of toluene, leading to changes in membrane fluidity.     

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.     

Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases

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Neuromuscular actions of endothelin on smooth, cardiac and skeletal muscle from guinea-pig, rat and rabbit

The peptide endothelin (human, porcine) was investigated for effects on basal muscle tone and on responses to transmural nerve stimulation in a series of smooth muscle preparations, as well as in guinea-pig atrium and rat and guinea-pig diaphragm. Endothelin lacked effect on basal tone or on spontaneous and electrically driven contractions in skeletal and atrial muscle. It contracted guinea-pig ileum, pulmonary and femoral arteries, rat anococcygeus, vas deferens and urinary bladder and rabbit taenia coli, whereas guinea-pig taenia was relaxed. Guinea-pig urinary bladder and vas deferens and rabbit iris sphincter were unaffected up to 3 x 10(-8) M. Endothelin thus has a unique pattern of smooth muscle effects, exhibiting mostly contractile but also relaxing effects. Endothelin modified contractile responses to transmural nerve stimulation, yielding marked and persistent enhancement, in guinea-pig and rat vas deferens, and enhancement also in guinea-pig pulmonary artery. In guinea-pig and rat vas deferens the response to exogenous ATP was increased by endothelin, thus suggesting a strong post-junctional enhancement of neurotransmission. In guinea-pig ileum nerve-induced responses were inhibited by endothelin, whereas exogeneous acetylcholine was enhanced, an effect suggesting a simultaneous pre-junctional inhibition and post-junctional enhancement. The Ca2+ channel blocker felodipine counteracted the stimulatory effects of endothelin on tone and transmurally induced contractions. Tachyphylaxis to endothelin action was sometimes evident, but the anococcygeus being less prone to this might be useful for studies on endothelin antagonism. Endothelin thus has prominent post-junctional, and also probably pre-junctional, effects, lending further support for a distinct biological role of this peptide.