Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function

Niemann-Pick type C1 (NPC1) disease is a fatal hereditary disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. Although the NPC1 gene has been identified, the molecular mechanism responsible for neuronal dysfunction in brains of patients with NPC1 disease remains unknown. This study demonstrates that the amount of cholesterol within mitochondria membranes is significantly elevated in NPC1 mouse brains and neural cells. In addition, the mitochondrial membrane potential, the activity of ATP synthase, and henceforth the level of ATP are markedly decreased in NPC1 mouse brains and neurons. Importantly, reducing the level of cholesterol within mitochondrial membranes using methyl-beta-cyclodextrin can restore the activity of ATP synthase. Finally, NPC1 neurons show an impaired neurite outgrowth, which can be rescued by exogenous ATP. These results suggest that mitochondrial dysfunctions and subsequent ATP deficiency, which are induced by altered cholesterol metabolism in mitochondria, may be responsible for neuronal impairment in NPC1 disease.     

Bradykinin microinjection in the paratrigeminal nucleus triggers neuronal discharge in the rat rostroventrolateral reticular nucleus

A small collection of neurons in the dorsal lateral medulla, the paratrigeminal nucleus (Pa5), projects directly to the rostroventrolateral reticular nucleus (RVL). Bradykinin (BK) microinjections in the Pa5 produce marked pressor responses. Also, the Pa5 is believed to be a component of the neuronal substrates of the somatosensory response and the baroreflex arc. Considering the developing interest in the functional physiology of the Pa5, the present study was designed to characterize RVL neuronal activity in response to BK microinjections in the Pa5 as well as to phenylephrine-induced blood pressure increases in freely behaving rats. Of the 46 discriminated RVL neurons, 82% responded with a 180% mean increase in firing rate after BK application to the paratrigeminal nucleus, before the onset of the blood pressure increase. Thirty (79%) of the RVL BK-excited neurons were baroreceptor-inhibited units that responded with a 30% decrease in firing rate in response to a phenylephrine-produced increase of blood pressure. Twenty-seven (71%) units of the latter population displayed cardiac-cycle-locked rhythmic activity. The findings demonstrate a BK-stimulated functional connection between the Pa5 and RVL that may represent the neural pathway in the BK-mediated pressor response. This pathway may be relevant to baroreflex mechanisms since it relates to cardiovascular pressure-sensitive neurons.     

The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptosis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of the FADD death effector domain. E6 is produced in two forms as follows: a full-length version of approximately 16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.     

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) alpha subunits alone. Because native BKCa channel complexes may include the auxiliary subunit beta1, we investigated whether beta1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with beta1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75 mV) as compared with that with alpha alone (-30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of beta1. Oxidation of cysteine and methionine residues within beta1 was not involved in these potentiated effects because expression of mutant beta1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type beta1. Unlike the results with alpha alone, oxidation by Ch-T caused a significant acceleration of channel activation only when beta1 was present. The beta1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming alpha subunit are greatly amplified by the presence of beta1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within beta1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with beta1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.     

Induction of autoantibodies to CCR5 in macaques and subsequent effects upon challenge with an R5-tropic simian/human immunodeficiency virus

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV(SF162P3) in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV(SF162P3). Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.     

IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.     

Bioconjugatable porphyrins bearing a compact swallowtail motif for water solubility

A broad range of applications requires access to water-soluble, bioconjugatable porphyrins. Branched alkyl groups attached at the branching site to the porphyrin meso position are known to impart high organic solubility. Such "swallowtail" motifs bearing a polar group (hydroxy, dihydroxyphosphoryl, dihydroxyphosphoryloxy) at the terminus of each branch have now been incorporated at a meso site in trans-AB-porphyrins. The incorporation of the swallowtail motif relies on rational synthetic methods whereby a 1,9-bis(N-propylimino)dipyrromethane (bearing a bioconjugatable tether at the 5-position) is condensed with a dipyrromethane (bearing a protected 1,5-dihydroxypent-3-yl unit at the 5-position). The two hydroxy groups in the swallowtail motif of each of the resulting zinc porphyrins can be transformed to the corresponding diphosphate or diphosphonate product. A 4-(carboxymethyloxy)phenyl group provides the bioconjugatable tether. The six such porphyrins reported here are highly water-soluble (> or =20 mM at room temperature in water at pH 7) as determined by visual inspection, UV-vis absorption spectroscopy, or 1H NMR spectroscopy. Covalent attachment was carried out in aqueous solution with the unprotected porphyrin diphosphonate and a monoclonal antibody against the T-cell receptor CD3epsilon. The resulting conjugate performed comparably to a commercially available fluorescein isothiocyanate-labeled antibody with Jurkat cells in flow cytometry and fluorescence microscopy assays. Taken together, this work enables preparation of useful quantities of water-soluble, bioconjugatable porphyrins in a compact architecture for applications in the life sciences.