Systematic Analysis of Intracellular Trafficking Motifs Located within the Cytoplasmic Domain of Simian Immunodeficiency Virus Glycoprotein gp41

Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Protein kinase C is involved in laminin stimulation of neurite outgrowth

We are investigating the intracellular events involved in the induction of neurite outgrowth. The phorbol ester TPA, an activator of protein kinase C, potentiates neurite outgrowth from ciliary ganglion neurons cultured on suboptimal laminin concentrations, but not on optimal laminin concentrations. TPA also stimulates growth on fibronectin and collagen similar to that observed on laminin under control conditions. Manipulations that elevate intracellular cAMP levels (expected to activate A kinase) reduce neurite outgrowth on laminin. The protein kinase C inhibitors H7 and sphingosine inhibit neurite outgrowth on laminin in a reversible and dose-dependent manner. H7 does not inhibit the process outgrowth induced by concanavalin A in the same neurons. The results suggest that activation of protein kinase C is an important step in the neurite outgrowth caused by laminin binding to its receptor(s).     

Transgenic expression of the viral FLIP MC159 causes lpr/gld-like lymphoproliferation and autoimmunity

Death receptor-induced programmed cell death (PCD) is crucial for the maintenance of immune homeostasis. However, interference of downstream death receptor signaling by genetic ablation or transgenic (Tg) expression of different apoptosis inhibitors often impairs lymphocyte activation. The viral FLICE (caspase-8)-like inhibitor proteins (v-FLIPs) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. We generated Tg mice expressing the v-FLIP MC159 from Molluscum contagiosum virus under the control of the H2Kb class I MHC promoter to examine the role of death receptor-induced PCD in the control of immune functions and homeostasis. We found that expression of MC159 led to lymphoproliferation and autoimmunity as exemplified by T and B lymphocyte expansion, accumulation of TCRalphabeta+ CD3+ B220+ CD4- CD8- lymphocytes in secondary lymphoid organs, elevated serum Ig levels, and increased anti-dsDNA Ab titers. These phenotypes were caused by defective death receptor-induced apoptosis, but not by defective passive cell death in the absence of mitogenic stimulation. Lymphocyte activation was normal, as demonstrated by normal thymidine incorporation and CSFE dilution of T cells stimulated with anti-CD3 and anti-CD28 Abs. In addition, effector CD8+ T cell responses to acute and memory lymphocytic choriomeningitis virus infections were unaffected in the Tg mice. These phenotypes are reminiscent of the lpr and gld mice, and show that the v-FLIP MC159 is a bona fide PCD inhibitor that does not interfere with other essential lymphocyte functions. Thus, the MC159-Tg mice provide a model to study the effects of PCD in immune responses without hampering other important lymphocyte functions.     

Comparison of laser Doppler imaging, fingertip lacticemy test, and nailfold capillaroscopy for assessment of digital microcirculation in systemic sclerosis

Introduction  

Laser Doppler imaging (LDI) is a relatively new method for assessing the functional aspect of superficial skin blood flow in systemic sclerosis (SSc) and Raynaud's phenomenon. The present study investigated the dynamic behavior of digital skin microvascular blood flow before and after cold stimulus (CS) in SSc patients and in healthy controls by means of a comprehensive approach of the functional (LDI), morphological (nailfold capillaroscopy (NFC)), and biochemical (fingertip lacticemy (FTL)) microcirculation components.     

Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.      

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.