COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Dual action of anti-sporozoite antibodies in vitro

With the use of a double staining technique that permits localization of the sporozoite during the process of entering a host cell, we studied the biologic effects of three mAb directed against determinants contained in the circumsporozoite of Plasmodium yoelii. These mAb, which included one IgM and two IgG3, were studied in primary cultures of rodent hepatocytes inoculated with sporozoites of P. yoelii. These results confirm previous reports of the extended action of antibodies on Plasmodium falciparum after entering hepatocytes by producing a strong intrahepatocyte inhibitory effect in addition to the inhibitory effect on sporozoite entry. As with P. falciparum the intracellular effects on P. yoelii liver stages are only observed when the antibodies are present at the time the sporozoite enters the cell. While carrying out experiments on this phenomenon, it was discovered that, at lowered antibody concentrations, an increase in number of maturing liver schizonts occurs, with the increase or enhancement of infection reaching up to 150% of that of controls. It was also observed that there was an inverse relationship between the antibody concentration that was inhibitory and that which enhanced parasite infectivity.     

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.     

Tumor-based rhythms of anticancer efficacy in experimental models

Experimental tumor models constitute a prerequisite toward chronotherapy testing in cancer patients. Studies in experimental models are required to understand the relation between tumor rhythms and antitumor treatments efficacy. In healthy tissues, cell proliferation, and differentiation processes are regulated precisely and exhibit marked circadian rhythmicity. Experimental and human tumors can retain circadian rhythms or display altered oscillations. Healthy tissues can also display rhythm modifications, possibly related to cancer stage. Cellular rhythms modulate the metabolism of cytotoxic agents and the cellular response to them; hence, they determine the chronopharmacology of anticancer drugs. Circadian rhythms in host tolerability and/or cancer chemotherapy efficacy have been demonstrated with nontoxic doses of drugs in several experimental tumor models, while in other ones a circadian-time effect was only seen within a specific dose range. The usual coupling between tolerability and efficacy rhythms of anticancer agents has resulted in significant improvement of their therapeutic index. Results of laboratory animal studies have been extrapolated to the design of clinical cancer therapy trials involving a chronobiological approach.     

Observation of protein-derived (BSA) oxygen-centered radicals by EPR spin-trapping techniques

Electron paramagnetic resonance (EPR) spin-trapping experiments, employing the novel spin-trap DEPMPO, provide evidence for the formation of protein-peroxyl radicals from the reaction of bovine serum albumin (BSA) or lysozyme with HO · in the presence of O 2 . Spin-trapping leads to the detection of anisotropic spectra of partially immobilized protein-peroxyl spin-adducts; positive identification is based on a novel spectrum simulation approach (through which broadened anisotropic spectra are simulated and compared with experiment) and by comparison of results with those obtained when MeO 2 · is trapped and the adduct frozen in a solid matrix.     

Distribution of intraventricularly injected horseradish peroxidase in cerebrospinal fluid compartments of the rat spinal cord

Summary The circulation of the cerebrospinal fluid along the central canal and its access to the parenchyma of the spinal cord of the rat have been analyzed by injection of horseradish peroxidase (HRP) into the lateral ventricle. Peroxidase was found throughout the central canal 13 min after injection, suggesting a rapid circulation of cerebrospinal fluid along the central canal of the rat spinal cord. It was cleared from the central canal within 2 h, in contrast with the situation in the brain tissue, where it remained in the periventricular areas for 4 h. In the central canal, HRP bound to Reissner's fiber and the luminal surface of the ependymal cells; it penetrated through the intercellular space of the ependymal lining, reached the subependymal neuropil, the basement membrane of local capillaries, and appeared in the lumen of endothelial pinocytotic vesicles. Furthermore, it accumulated in the labyrinths of the basement membrane contacting the basolateral aspect of the ependymal cells. In ependymocytes, HRP was found in single pinocytotic vesicles. The blood vessels supplying the spinal cord were classified into two types. Type-A vessels penetrated the spinal cord laterally and dorsally and displayed the tracer along their external wall as far as the gray matter. Type-B vessels intruded into the spinal cord from the medial ventral sulcus and occupied the anterior commissure of the gray matter, approaching the central canal. They represented the only vessels marked by HRP along their course through the gray matter. HRP spread from the wall of type-B vessels, labeling the labyrinths, the intercellular space of the ependymal lining, and the lumen of the central canal. This suggests a communication between the central canal and the outer cerebrospinal fluid space, at the level of the medial ventral sulcus, via the intercellular spaces, the perivascular basement membrane and its labyrinthine extensions.