Plasmacytoid dendritic cell-derived IFNα modulates Th17 differentiation during early Bordetella pertussis infection in mice.

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis). T helper 17 (Th17) cells have a central role in the resolution of the infection. Emerging studies document that type I interferons (IFNs) suppress Th17 differentiation and interleukin (IL)-17 responses in models of infection and chronic inflammation. As plasmacytoid dendritic cells (pDCs) are a major source of type I IFNs, we hypothesize that during B. pertussis infection in mice, pDC-derived IFNα inhibits a rapid increase in Th17 cells. We found that IFNα-secreting pDCs appear in the lungs during the early stages of infection, while a robust rise of Th17 cells in the lungs is detected at 15 days post-infection or later. The presence of IFNα led to reduced Th17 differentiation and proliferation in vitro. Furthermore, in vivo blocking of IFNα produced by pDCs during infection with B. pertussis infection resulted in early increase of Th17 frequency, inflammation, and reduced bacterial loads in the airways of infected mice. Taken together, the experiments reported here describe an inhibitory role for pDCs and pDC-derived IFNα in modulating Th17 responses during the early stages of B. pertussis infection, which may explain the prolonged nature of whooping cough.

Proinflammatory and proapoptotic activities associated with Bordetella pertussis filamentous hemagglutinin.

Filamentous hemagglutinin (FHA) is a dominant cell surface-associated Bordetella pertussis adhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other activities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-type B. pertussis resulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-alpha), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-alpha monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-alpha receptor 1 inhibited FHA-associated apoptosis by 47.7% (P = 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in the B. pertussis-host interaction.

Light chain-dependent regulation of Kinesin's interaction with microtubules.

We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates.

BACKGROUND: Cytoplasmic mRNA localization is a widespread mechanism for restricting the translation of specific mRNAs to distinct regions of eucaryotic cells. This process involves specific interactions between cellular factors and localization signals in the 3' untranslated regions of the localized mRNA. Because only a few of these cellular factors have been identified, it is not known whether common factors are utilized for the localization of different mRNAs. We recently discovered Vera, a protein that binds specifically to the Vg1 localization element and is involved in the localization of Vg1 mRNA in Xenopus oocytes. RESULTS: To characterize further the role of Vera in the localization of Vg1 mRNA, we have purified the Vera protein and cloned its gene. Vera is homologous to chicken zip-code-binding protein (ZBP), which binds to a short RNA sequence required for localization of beta-actin mRNA in chick embryo fibroblasts. The predicted amino-acid sequences of Vera and ZBP contain five RNA-binding domains and putative signals for nuclear localization and export. Like the binding of ZBP to beta-actin mRNA, Vera specifically binds to a repeated sequence motif in the Vg1 localization element that is required for Vg1 mRNA localization in Xenopus oocytes. CONCLUSIONS: Vera, a highly conserved component of the mRNA localization machinery, participates in localizing different mRNAs in different cell types. Thus, Vera appears to be a general factor for mRNA localization, and additional factors may be required to specify diverse patterns of RNA localization.

KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

Transfer of the scorpion toxin receptor to an insensitive potassium channel.

Voltage-dependent potassium channels belong to a family of structurally related cation channels that underlie the electrical activity of excitable cells. Many potassium channels are blocked with high affinity by scorpion toxins, whereas others are completely insensitive. We transferred toxin sensitivity from the highly sensitive Kv1.3 (KV3) to the insensitive Kv2.1 (DRK1) potassium channel by transferring the stretch of amino acids between transmembrane domains 5/6. We provide evidence that this S5-S6 linker, which has been shown to comprise the pore-forming region, is probably the only part of the ion channel that directly interacts with bound toxin. Using site-directed mutagenesis, we identified specific residues in the S5-S6 linker that are responsible for the acquisition of toxin sensitivity by Kv2.1.

Mutations in the K+ channel signature sequence.

Potassium channels share a highly conserved stretch of eight amino acids, a K+ channel signature sequence. The conserved sequence falls within the previously defined P-region of voltage-activated K+ channels. In this study we investigate the effect of mutations in the signature sequence of the Shaker channel on K+ selectivity determined under bi-ionic conditions. Nonconservative substitutions of two threonine residues and the tyrosine residue leave selectivity intact. In contrast, mutations at some positions render the channel nonselective among monovalent cations. These findings are consistent with a proposal that the signature sequence contributes to a selectivity filter. Furthermore, the results illustrate that the hydroxyl groups at the third and fourth positions, and the aromatic group at position seven, are not essential in determining K+ selectivity.

Effect of counselor training on skills development and psychosocial status of volunteers with systemic lupus erythematosus.

Volunteers with systemic lupus erythematosus were recruited for a telephone service to provide psychosocial support to peers. The volunteers attended an 8-week counselor training program. The aim was to evaluate the impact of the training on counselors' skill development and to record possible changes in the counselors' psychosocial status. A second group of trainees (n = 15) was used as a delayed control for the first group (n = 14). The data were analyzed using multivariate repeated measures analysis of variance. Tests chosen to monitor psychosocial status included: Arthritis Impact Measurement Scales Anxiety and Depression subscales, Arthritis Helplessness Index, Wallston General Self-Efficacy Scale, University of California at Los Angeles Loneliness Scale, Rosenberg Self-Esteem, and Campbell Personal Competence scales. Tests selected to measure skills and knowledge.

A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels.

The overall sequence similarity between the voltage-activated K+ channels and cyclic nucleotide-gated ion channels from retinal and olfactory neurons suggests that they arose from a common ancestor. On the basis of sequence comparisons, mutations were introduced into the pore of a voltage-activated K+ channel. These mutations confer the essential features of ion conduction in the cyclic nucleotide-gated ion channels; the mutant K+ channels display little selectivity among monovalent cations and are blocked by divalent cations. The property of K+ selectivity is related to the presence of two amino acids that are absent from the pore-forming region of the cyclic nucleotide-gated channels. These data demonstrate that very small differences in the primary structure of an ion channel can account for extreme functional diversity, and they suggest a possible connection between the pore-forming regions of K+, Ca2+, and cyclic nucleotide-gated ion channels.

Gonadotropin-releasing hormone specific binding sites in normal and malignant renal tissue.

Renal cell carcinoma has been reported to contain estrogen and progesterone receptors. Thus, it has been suggested that these tumors are hormone dependent in a similar manner described for the breast and prostate cancers. It has been recently shown that mammary and prostate tumor cells contain gonadotropin-releasing hormone (GnRH) receptors and are growth inhibited directly by GnRH antagonists. In this study we examined for the presence of GnRH, estrogen and progesterone receptors in normal and malignant renal tissues. Estrogen receptors were found both in the normal and malignant kidney while progesterone receptors were present only in the normal tissue. Specific binding of [125I]buserelin, a GnRH agonist, was evident in renal carcinoma and in normal kidney and was displaced with equal efficiency by unlabeled buserelin and by D-Trp6-GnRH, but not by unrelated peptides such as thyrotropin releasing hormone and oxytocin. The non-linear scatchard curve obtained for buserelin binding, suggests the presence of at least two binding sites, one with high affinity in the nanomolar range and another in the micromolar range.

Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors.

We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.

Academic behavior and grades of mainstreamed students with mild disabilities.

This study examined the relationship between the classroom behaviors and final grades of mainstreamed students with mild disabilities in three urban schools in Grades 3-9. Significant correlations were found for all academic behaviors except attendance and homework at the elementary level and written work at the junior high level. In addition, significant differences in reading scores were found between mainstreamed and regular elementary school students. Findings indicate that mainstreamed students with mild disabilities may participate in academic lessons as well or nearly as well as their peers without disabilities.

Direct effects of luteinizing hormone-releasing hormone agonists and antagonists on MCF-7 mammary cancer cells.

The binding of luteinizing hormone-releasing hormone (LH-RH) analogues to the human mammary tumor cell line MCF-7 and their effect on the cell proliferation was studied to elucidate their direct action on estrogen-dependent mammary tumors. The growth rate of these cells was doubled by the addition of 1 nM estradiol to cells maintained in an estrogen-deficient medium. Although the basal growth rate was only slightly inhibited by the LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), the estrogen-stimulated growth was completely abolished by the antagonist. In contrast, the LH-RH agonist buserelin stimulated cell growth in estrogen-deficient medium, whereas it had no effect in the presence of estrogen. 125I-labeled buserelin was used for the measurement of LH-RH receptors on MCF-7 cells. A Scatchard plot analysis of buserelin-specific binding revealed a nonlinear plot, which suggested the presence of one high-affinity binding site with a Kd of 1.4 +/- 1.0 nM and the remaining sites with low affinity (Kd = 1.3 +/- 1.0 microM). The binding of 125I-labeled buserelin was displaced equally well by unlabeled buserelin and by the LH-RH antagonist SB-75, suggesting that both analogues are bound to the same receptor. When parallel experiments were performed with 125I-labeled SB-75, the binding was displaced by unlabeled SB-75 and other antagonists, but only partially displaced by unlabeled buserelin. The results suggest that in these mammary tumor cells there is a LH-RH antagonist binding site that is not recognizable by LH-RH agonists. This hypothesis was tested by measuring cell growth in the presence of both agonists and antagonists. It was found that SB-75 inhibited the stimulation of growth by buserelin, but buserelin did not prevent the inhibition by the antagonist of the estrogen-dependent growth. These results suggest that antagonists directly inhibit mammary tumor growth, not only by competing with LH-RH high-affinity receptors, but also by other mechanisms mediated by low-affinity antagonist binding sites.

Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel.

The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor.

The Shaker K+ channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K+ channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K+ channels and may contribute to the formation of the ion conduction pore.