Total ear reconstruction in the devascularized temporoparietal region: I. Use of the contralateral temporoparietal fascial free flap

Total ear reconstruction by the use of contralateral temporoparietal fascial free flap and autogenous costal cartilage was performed in 16 patients presenting with a devascularized temporoparietal region resulting from trauma or prior surgery. The microsurgical success rate was 87.5 percent (14 of 16 transplants). On evaluation of the final aesthetic result in 11 patients followed up for more than 3 years, nine patients were graded good-to-excellent and two patients exhibited fair-to-poor results. Despite the relatively long operating hours and the comparatively low microsurgical success rate, ear reconstruction by autogenous tissue transplantation has proved to be an encouraging and worthwhile experience. This article presents the clinical cases and discusses the technical details.     

Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice

The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)     

Molecular cloning and characterization of Pals, proteins associated with mLin-7

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.     

Radiolabeling of paclitaxel with electrophilic 123I

123I-Labeled paclitaxel, [123I]-1 was prepared by electrophilic aromatic radioiodination of 3'-N-(p-trimethylstannylbenzoyl)-3'-debenzoylpaclitaxel 2 with 123I- in the presence of peracetic acid.     

Exercise training reverses cardiac aging phenotypes associated with heart failure with preserved ejection fraction in male mice

Heart failure with preserved ejection fraction (HFpEF) is the most common type of HF in older adults. Although no pharmacological therapy has yet improved survival in HFpEF, exercise training (ExT) has emerged as the most effective intervention to improving functional outcomes in this age‐related disease. The molecular mechanisms by which ExT induces its beneficial effects in HFpEF, however, remain largely unknown. Given the strong association between aging and HFpEF, we hypothesized that ExT might reverse cardiac aging phenotypes that contribute to HFpEF pathophysiology and additionally provide a platform for novel mechanistic and therapeutic discovery. Here, we show that aged (24–30 months) C57BL/6 male mice recapitulate many of the hallmark features of HFpEF, including preserved left ventricular ejection fraction, subclinical systolic dysfunction, diastolic dysfunction, impaired cardiac reserves, exercise intolerance, and pathologic cardiac hypertrophy. Similar to older humans, ExT in old mice improved exercise capacity, diastolic function, and contractile reserves, while reducing pulmonary congestion. Interestingly, RNAseq of explanted hearts showed that ExT did not significantly modulate biological pathways targeted by conventional HF medications. However, it reversed multiple age‐related pathways, including the global downregulation of cell cycle pathways seen in aged hearts, which was associated with increased capillary density, but no effects on cardiac mass or fibrosis. Taken together, these data demonstrate that the aged C57BL/6 male mouse is a valuable model for studying the role of aging biology in HFpEF pathophysiology, and provide a molecular framework for how ExT potentially reverses cardiac aging phenotypes in HFpEF.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

New fast BiFC plasmid assay system for in vivo protein-protein interactions.

In this age of massive genetic and protein information, a fast and reliable method of studying in vivo protein-protein interactions is necessary. We have developed a novel system that can overcome limitations of existing assay methods. This new method adopts two existing systems for fast analysis of diverse protein-protein interactions. For rapid, large-scale cloning, we adopted the Gateway system and developed novel destination vectors containing YFP N-terminus (YN) or YFP C-terminus (YC) to visualize protein-protein interactions in vivo using bimolecular fluorescence complementation (BiFC). Using this system, we investigated molecular interactions among the three POZ-domain regulatory proteins mAPM-1, LRF, KLHL10 that belong to a subgroup of human POZ-domain proteins, and showed that the POZ-domains of mAPM-1, LRF and KLHL10 could form both homodimers and heterodimers. This new method is a highly efficient, sensitive and specific assay method for protein-protein interaction in vivo.     

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate

Here, we present a simple method for controlling the density of Au nanoparticles (Au NPs) on a modified silicon substrate, by destabilizing the colloidal Au NPs with 3-mercaptopropyltrimethoxylsilane (3-MPTMS) for microelectromechanical-system-based applications to reduce tribological issues. A silicon surface was pretreated with a 3-MPTMS solution, immediately after which thiolated Au NPs were added to it, resulting in their uniform deposition on the silicon substrate. Without any material property change of the colloidal Au NPs, we observed the formation of large clusters Au NPs on the modified silicon surface. Analysis by scanning electron microscopy with energy dispersive X-ray spectroscopy indicated that the addition of 3-MPTMS resulted in an alternation of the chemical characteristics of the solution. Atomic force microscopy imaging supported the notion that silicon surface modification is the most important factor on tribological properties of materials along with ligand-modified Au NPs. The density of Au NPs on a silicon surface was significantly dependent on several factors, including the concentration of colloidal Au NPs, deposition time, and concentration of 3-MPTMS solution, while temperature range which was used throughout experiment was determined to have no significant effect. A relatively high density of Au NPs forms on the silicon surface as the concentrations of Au NPs and 3-MPTMS are increased. In addition, the maximum deposition of Au NPs on silicon wafer was observed at 3 h, while the effects of temperature variation were minimal.     

Moderate hypothermia attenuates α(1)-adrenoceptor-mediated contraction in isolated rat aorta: the role of the endothelium

Moderate hypothermia (25–31 °C) may have a significant influence on vascular tone. We investigated the cellular mechanisms by which moderate hypothermia alters α-adrenoceptor-mediated contraction in rat thoracic aortae. Cyclooxygenase inhibition by indomethacin; nitric oxide (NO) synthase inhibition by l-NAME; potassium channel and endothelium-derived hyperpolarizing factor (EDHF) inhibition by glibenclamide and TEA; G protein inhibition by pertussis toxin; α₂-adrenergic inhibition by yohimbine; and β-adrenergic inhibition by propranolol were assessed for their effect on the contractile response to the α1-adrenoceptor agonist phenylephrine (Phe) in combination with moderate hypothermia (25 °C). Moderate hypothermia produced a shift to the right for the Phe concentration–response curves in endothelium-intact (E+) and endothelium-denuded (E?) aortic rings. The maximal response to Phe in E+ rings was significantly decreased (P < 0.05) at 25 °C compared to 38 °C, whereas there was no significant difference in E? rings. Hypothermia-induced vasorelaxation in E+ rings was attenuated (P < 0.05) following combined pretreatment with l-NAME (10^?4 M) and indomethacin (10^?5 M), whereas other inhibitors had no significant effect. Importantly, the addition of TEA to rings that were pretreated with l-NAME and indomethacin exhibited no further attenuation (P > 0.05) of hypothermia-induced vasorelaxation. The concentrations of cGMP and cAMP, as measured by radioimmunoassay, were significantly increased (P < 0.05) in E+ rings at 25 °C compared to those at 38 °C, whereas there were no significant differences (P > 0.05) in E? rings. The present study demonstrated that rat aortic endothelium is stimulated during moderate hypothermia and that the NO–cGMP and prostacyclin (PGI₂)–cAMP pathways represent endothelium-dependent mechanisms of hypothermia-induced vasorelaxation. In contrast, EDHF may not be associated with hypothermia-induced vasorelaxation.     

Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis , all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm⁻²), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L⁻¹ protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L⁻¹) upon addition of polymyxin B nonapeptide at 50 and 20 mg L⁻¹, respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.