Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.     

Reverse-flow island sural flap

The reverse flow island sural flap is presented as an alternative to flaps currently used for reconstruction of small and medium substance losses in the distal third of the leg, ankle, and heel. This is a random type of flap, based on the reverse flow of the superficial sural artery, which mainly depends on the anatomy of the perforators of the peroneal artery system.The anatomic structures that constitute the pedicle are the superficial and deep fascias, the sural nerve, the short saphenous vein, and the superficial sural artery. The skin island and the subcutaneous cellular tissue complement the flap proper. This skin island was demarcated at any point of the median or distal thirds of the leg, having the short saphenous vein and the sural nerve on its central axis. The distal dissection limit of the pedicle is located 5 centimeters above the lateral malleolus. This limit is established so as to ensure the integrity of the perforators from the principal arteries of the leg, mainly the peroneal artery, responsible for the reverse flow nourishing the flap. These perforators will affect anastomoses with the superficial sural artery in charge of irrigating the structures compounding the flap.A total of 71 patients were operated on with this technique, some of them with basic pathologic abnormalities limiting the distal blood flow, such as diabetes mellitus, and some others having proven vascular insufficiency or displaying unstable areas attributable to problems such as pseudarthrosis and osteomyelitis, which needed to be covered. Fifteen flaps (21.1 percent) suffered partial necrosis, which did not compromise the final result, and another three (4.2 percent) showed total loss. The flap in question has great mobility and versatility, allowing the treatment of specific areas of the lower limb, without sacrificing important arteries or mobilizing structures that might bring about functional deficits.     

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.     

Nitric oxide inhibits irreversibly P815 cell proliferation: involvement of potassium channels

Abstract. Nitric oxide (NO) has been shown to inhibit both normal and cancer cell proliferation. Potassium channels are involved in cell proliferation and, as NO activates these channels, we investigated the effect of NO on the proliferation of murine mastocytoma cell lines and the putative involvement of potassium channels. NO (in the form of NO donors) caused dose-dependent inhibition of cell proliferation in the P815 cell line inducing growth arrest in the mitosis phase. Incubation with NO donor for 4 or 24 h had a similar inhibitory effect on cell proliferation, indicating that this effect is irreversible. The inhibitory effect of NO was completely prevented by the blockade of voltage- and calcium-dependent potassium channels, but not by blockade of ATP-dependent channels. NO inhibition of cell proliferation was unaffected by guanylate cyclase and by cytoskeleton disruptors. Therefore, NO inhibits cell proliferation irreversibly via a potassium channel-dependent but guanylate cyclase-independent pathway in murine mastocytoma cells.     

Molecular mechanisms of nickel carcinogenesis

A brief review of the molecular mechanisms of nickel carcinogenesis is presented. Molecular mechanisms of nickel carcinogenesis are considered from the point-of-view of nickel-induced gene silencing by DNA hypermethylation in mammalian cells and by its ability to inhibit histone acetylation. Model systems designed to study the molecular mechanism of gene silencing are discussed.     

Release of NO by a nitrosyl complex upon activation by the mitochondrial reducing power

The reaction of trans-[Ru(NH(3))(4)P(OEt)(3)NO](3+) and mitochondria was investigated through differential pulse polarography and fluorimetry. The nitrosyl complex undergoes one-electron reduction centered on the NO ligand site. The reaction between the mitochondrial reductor and trans-[Ru(NH(3))(4)P(OEt)(3)NO](3+) exhibits a second order specific rate constant calculated as k=2 x 10(1) M(-1) s(-1). The reduced species, trans-[Ru(NH(3))(4)P(OEt)(3)NO](2+), quickly releases NO, yielding trans-[Ru(NH(3))(4)P(OEt)(3)H(2)O](2+). The low toxicities of both trans-[Ru(NH(3))(4)P(OEt)(3)(NO)](2+) and trans-[Ru(NH(3))(4)P(OEt)(3)H(2)O](2+) and its ability to release NO after reductive activation in a biological medium make the nitrosyl compound a useful model of a hypotensive drug.     

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.