The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.     

Electron transfer oxidation of tryptophan and tyrosine by triplet states and oxidized radicals of flavin sensitizers: a laser flash photolysis study

Mitogen-activated protein (MAP) kinases are activated by dual-specificity kinases, termed MEKs. Using MEK2 as bait in yeast two-hybrid screening, besides c-Raf and KSR, A-Raf was identified as a novel partner that interacts with MEK2. This interaction was confirmed by in vitro binding assay. Further investigation indicates that regions critical for this interaction were located between residues 255 and 606 that represent the kinase domain of A-Raf.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like subunit and its role in DNA repair

We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.