No abstract

MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-translational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447-->Asp mutation was introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37 degrees C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/delta nmt, and nmt delta/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmt delta/nmtG447D strain required myristate for growth. This was true at both 24 and 37 degrees C. Palmitate could not substitute for myristate. Incubation of nmt delta/nmtG447D cells at 37 degrees C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 microM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/delta nmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmt delta/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.

No abstract

Annexin V recognizes apoptotic cells by specific molecular interaction with phosphatidyl serine, a lipid that is normally sequestered in the inner leaflet of the cell membrane, but is translocated to the outer leaflet in apoptotic cells, such as foam cells of atherosclerotic plaque. Annexin V could potentially deliver carried materials (such as superparamagnetic contrast agents for magnetic resonance imaging) to sites containing apoptotic cells, such as high grade atherosclerotic lesions, so we administered biochemically-derivatized (annexin V) superparmagnetic iron oxide particles (SPIONs) parenterally to two related rabbit models of human atherosclerosis. We observe development of negative magnetic resonance imaging (MRI) contrast in atheromatous lesions and but not in healthy artery. Vascular targeting by annexin V SPIONs is atheroma-specific (i.e., does not occur in healthy control rabbits) and requires active annexin V decorating the SPION surface. Targeted SPIONs produce negative contrast at doses that are 2,000-fold lower than reported for non-specific atheroma uptake of untargeted superparamagnetic nanoparticles in plaque in the same animal model. Occlusive and mural plaques are differentiable. While most of the dose accumulates in liver, spleen, kidneys and bladder, annexin V SPIONs also partition rapidly and deeply into early apoptotic foamy macrophages in plaque. Contrast in plaque decays within 2 months, allowing MRI images to be replicated with a subsequent, identical dose of annexin V SPIONs. Thus, biologically targeted superparamagnetic contrast agents can contribute to non-invasive evaluation of cardiovascular lesions by simultaneously extracting morphological and biochemical data from them.

No abstract

We demonstrate high sensitivity AlGaN/GaN field effect transistor biosensors with a control gate electrode for streptavidin detection. The device active area is functionalized with 3-Aminopropyltriethoxysilane and N-hydroxysulfosuccinimide-biotin for streptavidin binding. Without any electrochemical side effects, a gate voltage is applied through a Pt control electrode to the solution so that the device operates sensitively in the subthreshold regime. Due to the logarithmic relationship between the channel current and gate voltage in the subthreshold regime, at a concentration of 4.73 pM streptavidin, the device exhibits 9.97% current change in the subthreshold regime compared with the current in phosphate buffered saline solution. In the linear regime, the current change is 0.49% at the same streptavidin concentration.

Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) is a promising probe for biological electron paramagnetic resonance (EPR) oximetry and is being developed for clinical use. However, clinical applicability of LiNc-BuO may be hindered by potential limitations associated with biocompatibility,

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-nbutoxynaphthalo-cyanine (LiNc-BuO), enables repeated measurements of pO 2 from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNcBuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.